Kinase inhibitor

ABSTRACT

There is provided a compound of formula VIIa, 
                         
wherein Q x  represents —C(O)O—C 1-4  alkyl, or a salt or protected derivative thereof, and other compounds that are useful in the preparation of compounds that have antiinflammatory activity (e.g., through inhibition of one or more of members of: the family of p38 mitogen-activated protein kinase enzymes; Syk kinase; and members of the Src family of tyrosine kinases) and has use in therapy, including in pharmaceutical combinations, especially in the treatment of inflammatory diseases, including inflammatory diseases of the lung, eye and intestines.

FIELD OF THE INVENTION

This invention relates, inter alia, to a compound which is anantiinflammatory agent (e.g. through inhibition of one or more ofmembers of: the family of p38 mitogen-activated protein kinase enzymes(referred to herein as p38 MAP kinase inhibitors), for example the alphakinase sub-type thereof; Syk kinase; and the Src family of tyrosinekinases). The invention also relates to the use of this compound intherapy, including in mono- and combination therapies, especially in thetreatment of inflammatory diseases, including inflammatory diseases ofthe lung (such as asthma and chronic obstructive pulmonary disease(COPD)), eye (such as uveitis or keratoconjunctivitis sicca (dry eyedisease, also known as xerophthalmia)) and gastrointestinal tract (suchas Crohn's disease and ulcerative colitis).

BACKGROUND OF THE INVENTION

The listing or discussion of an apparently prior-published document inthis specification should not necessarily be taken as an acknowledgementthat the document is part of the state of the art or is common generalknowledge.

Four p38 MAPK isoforms (alpha, beta, gamma and delta respectively) havebeen identified, each displaying different patterns of tissueexpression. The p38 MAPK alpha and beta isoforms are found ubiquitouslythroughout the body, are present in many different cell types and areinhibited by a number of previously described small molecular weightcompounds. Early classes of inhibitors were highly toxic due to thebroad tissue distribution of these isoforms which resulted in off-targeteffects of the compounds. Some of the more recently identifiedinhibitors show improved selectivity for p38 MAPK alpha and betaisoforms and have wider safety margins.

p38 MAP kinase is believed to play a pivotal role in many of thesignalling pathways that are involved in initiating and maintainingchronic, persistent inflammation in human disease, for example, insevere asthma, COPD and inflammatory bowel disease (IBD). There is nowan abundant literature which demonstrates that p38 MAP kinase isactivated by a range of pro-inflammatory cytokines and that itsactivation results in the recruitment and release of furtherpro-inflammatory cytokines. Indeed, data from some clinical studiesdemonstrate beneficial changes in disease activity in patients duringtreatment with p38 MAP kinase inhibitors. For instance, Smith describesthe inhibitory effect of p38 MAP kinase inhibitors on TNFα (but notIL-8) release from human PBMCs (Smith, S. J., Br. J. Pharmacol., 2006,149:393-404).

The use of inhibitors of p38 MAP kinase in the treatment of COPD and IBDhas also been proposed. Small molecule inhibitors targeted to p38MAPKα/β have proved to be effective in reducing various parameters ofinflammation in:

-   -   cells and tissues obtained from patients with COPD, who are        generally corticosteroid insensitive (Smith, S. J., Br. J.        Pharmacol., 2006, 149:393-404);    -   biopsies from IBD patients (Docena, G. et al., J. Trans.        Immunol., 2010, 162:108-115); and    -   in vivo animal models (Underwood, D. C. et al., Am. J. Physiol.,        2000, 2791895-902; Nath, P. et al., Eur. J. Pharmacol., 2006,        544:160-167).

Irusen and colleagues also suggested the possibility of involvement ofp38 MAPKα/β on corticosteroid insensitivity via the reduction of bindingaffinity of the glucocorticoid receptor (GR) in nuclei (Irusen, E. etal., J. Allergy Clin. Immunol., 2002, 109:649-657). Clinicalinvestigations in inflammatory diseases with a range of p38 MAP kinaseinhibitors, including AMG548, BIRB 796, VX702, SCIO469 and SCIO323, havebeen described (Lee, M. R. and Dominguez, C., Current Med. Chem., 2005,12:2979-2994.). However, the major obstacle hindering the utility of p38MAP kinase inhibitors in the treatment of human chronic inflammatorydiseases has been the toxicity observed in patients. This has beensufficiently severe to result in the withdrawal from clinicaldevelopment of many of the compounds progressed, including all thosespecifically mentioned above.

COPD is a condition in which the underlying inflammation is reported tobe substantially resistant to the anti-inflammatory effects of inhaledcorticosteroids. Consequently, a superior strategy for treating COPDwould be to develop an intervention which has both inherentanti-inflammatory effects and the ability to increase the sensitivity ofthe lung tissues of COPD patients to inhaled corticosteroids. The recentpublication of Mercado et al. (2007; American Thoracic Society AbstractA56) demonstrates that silencing p38 MAPK γ has the potential to restoresensitivity to corticosteroids. Thus, there may be a dual benefit forpatients in the use of a p38 MAP kinase inhibitor for the treatment ofCOPD.

Many patients diagnosed with asthma or with COPD continue to suffer fromuncontrolled symptoms and from exacerbations of their medical conditionthat can result in hospitalisation. This occurs despite the use of themost advanced, currently available treatment regimens, comprising ofcombination products of an inhaled corticosteroid and a long actingβ-agonist. Data accumulated over the last decade indicates that afailure to manage effectively the underlying inflammatory component ofthe disease in the lung is the most likely reason that exacerbationsoccur. Given the established efficacy of corticosteroids asanti-inflammatory agents and, in particular, of inhaled corticosteroidsin the treatment of asthma, these findings have provoked intenseinvestigation. Resulting studies have identified that some environmentalinsults invoke corticosteroid-insensitive inflammatory changes inpatients' lungs. An example is the response arising fromvirally-mediated upper respiratory tract infections (URTI), which haveparticular significance in increasing morbidity associated with asthmaand COPD.

It has been disclosed previously that compounds that inhibit theactivity of both the c-Src and Syk kinases are effective agents againstrhinovirus replication (Charron, C. E. et al., WO 2011/158042) and thatcompounds that inhibit p59-HCK are effective against influenza virusreplication (Charron, C. E. et al., WO 2011/070369). Taken together withinhibition of p38 MAPK, these are particularly attractive properties forcompounds to possess that are intended to treat patients with chronicrespiratory diseases.

Certain p38 MAPK inhibitors have also been described as inhibitors ofreplication of respiratory syncytial virus (Cass L. et al., WO2011/158039).

The precise etiology of IBD is uncertain, but is believed to be governedby genetic and environmental factors that interact to promote anexcessive and poorly controlled mucosal inflammatory response directedagainst components of the luminal microflora. This response is mediatedthrough infiltration of inflammatory neutrophils, dendritic cells andT-cells from the periphery. p38 has become an obvious target forinvestigation in IBD models as a consequence of its ubiquitousexpression in inflammatory cells. Studies investigating the efficacy ofp38 inhibitors in animal models of IBD and human biopsies from IBDpatients indicated that p38 could be a target for the treatment of IBD(Hove, T. ten et al., Gut, 2002, 50:507-512, Docena, G. et al., J.Trans. Immunol, 2010, 162:108-115). However, these findings are notcompletely consistent with other groups reporting no effect with p38inhibitors (Malamut G. et al., Dig. Dis. Sci, 2006, 51:1443-1453). Aclinical study in Crohn's patients using the p38 alpha inhibitor BIRB796demonstrated potential clinical benefit with an improvement inC-reactive protein levels. However this improvement was transient,returning to baseline by week 8 (Schreiber, S. et al., Clin. Gastro.Hepatology, 2006, 4:325-334). A small clinical study investigating theefficacy of CNI-1493, a p38 and Jnk inhibitor, in patients with severeCrohn's disease showed significant improvement in clinical score over 8weeks (Hommes, D. et al. Gastroenterology. 2002 122:7-14).

T cells are known to play a key role in mediating inflammation of thegastrointestinal tract. Pioneering work by Powrie and colleaguesdemonstrated that transfer of naive CD4+ cells into severely compromisedimmunodeficient (SCID) animals results in the development of colitiswhich is dependent on the presence of commensal bacteria (Powrie F. etal. Int Immunol. 1993 5:1461-71). Furthermore, investigation of mucosalmembranes from IBD patients showed an upregulation of CD4+ cells whichwere either Th1 (IFNg/IL-2) or Th2 (IL5/TGFb) biased depending onwhether the patient had Crohn's disease or ulcerative colitis (Fuss I J.et al. J Immunol. 1996 157:1261-70.). Similarly, T cells are known toplay a key role in inflammatory disorders of the eye with severalstudies reporting increased levels of T cell associated cytokines (IL-17and IL-23) in sera of Be

hets patients (Chi W. et al. Invest Ophthalmol Vis Sci. 200849:3058-64). In support of these observations, Direskeneli andcolleagues demonstrated that Be□hets patients have increased Th17 cellsand decreased Treg cells in their peripheral blood (Direskeneli H. etal. J Allergy Olin Immunol. 2011 128:665-6).

One approach to inhibit T cell activation is to target kinases which areinvolved in activation of the T cell receptor signalling complex. Sykand Src family kinases are known to play a key role in this pathway,where Src family kinases, Fyn and Lck, are the first signallingmolecules to be activated downstream of the T cell receptor (Barber E K.et al. PNAS 1989, 86:3277-81). They initiate the tyrosinephosphorylation of the T cell receptor leading to the recruitment of theSyk family kinase, ZAP-70. Animal studies have shown that ZAP-70knockout results in a SCID phenotype (Chan A C. et al. Science. 1994,10; 264(5165):1599-601).

A clinical trial in rheumatoid arthritis patients with the Syk inhibitorFostamatinib demonstrated the potential of Syk as an anti-inflammatorytarget with patients showing improved clinical outcome and reduced serumlevels of IL-6 and MMP-3 (Weinblatt M E. et al. Arthritis Rheum. 200858:3309-18). Syk kinase is widely expressed in cells of thehematopoietic system, most notably in B cells and mature T cells.Through interaction with immunoreceptor tyrosine-based activation motifs(ITAM), it plays an important role in regulating T cell and B cellexpansion as well as mediating immune-receptor signalling ininflammatory cells. Syk activation leads to IL-6 and MMPrelease-inflammatory mediators commonly found upregulated ininflammatory disorders including IBD and rheumatoid arthritis (Wang Y D.et al World J Gastroenterol 2007; 13: 5926-5932, Litinsky I et al.Cytokine. 2006 January 33:106-10).

In addition to playing key roles in cell signalling events which controlthe activity of pro-inflammatory pathways, kinase enzymes are now alsorecognised to regulate the activity of a range of cellular functions,including the maintenance of DNA integrity (Shilo, Y. Nature ReviewsCancer, 2003, 3: 155-168) and co-ordination of the complex processes ofcell division. Indeed, certain kinase inhibitors (the so-called“Olaharski kinases”) have been found to alter the frequency ofmicronucleus formation in vitro (Olaharski, A. J. et al., PLoS Comput.Biol., 2009, 5(7), e1000446; doi: 10.1371/journal.pcbi.1000446).Micronucleus formation is implicated in, or associated with, disruptionof mitotic processes and is therefore undesirable. Inhibition ofglycogen synthase kinase 3α (GSK3α) was found to be a particularlysignificant factor that increases the likelihood of a kinase inhibitorpromoting micronucleus formation. Also, inhibition of the kinase GSK3βwith RNAi has been reported to promote micronucleus formation (Tighe, A.et al., BMC Cell Biology, 2007, 8:34).

Whilst it may be possible to attenuate the adverse effects of inhibitionof Olaharski kinases such as GSK3α by optimisation of the dose and/or bychanging the route of administration of a molecule, it would beadvantageous to identify further therapeutically useful molecules withlow or negligible inhibition of Olaharski kinases, such as GSK 3α and/orhave low or negligible disruption of mitotic processes (e.g. as measuredin a mitosis assay).

Various compounds, including urea derivatives, are disclosed asinhibiting one or more kinases. Examples of such compounds may be foundin WO 99/23091, WO 00/041698, WO 00/043384, WO 00/055139, WO 01/36403,WO 01/4115, WO 02/083628, WO 02/083642, WO 02/092576, WO 02/096876, WO2003/005999, WO 2003/068223, WO 2003/068228, WO 2003/072569, WO2004/014870, WO 2004/113352, WO 2005/005396, WO 2005/018624, WO2005/023761, WO 2005/044825, WO 2006/015775, WO 2006/043090, WO2007/004749, WO 2007/053394, WO 2013/050756, WO 2013/050757, WO2014/027209, WO 2014/033446, WO 2014/033447, WO 2014/033448, WO2014/033449, WO 2014/076484, WO 2014/140582 WO 2014/162121, WO2014/162122, WO 2014/162126 and WO 2015/092423. Further examples may befound in articles published in:

-   -   Curr. Opin. Drug Devel. (2004, 7(5), 600-616);    -   J. Med. Chem. (2007, 50, 4016-4026; 2009, 52, 3881-3891; and        2010, 53, 5639-5655);    -   Bioorg. Med. Chem. Lett. (2007, 17, 354-357; 2008, 18,        3251-3255; 2009, 19, 2386-2391; and 2010, 20, 4819-4824);    -   Curr. Top. Med. Chem. (2008, 8, 1452-1467);    -   Bioorg. Med. Chem. (2010, 18, 5738-5748);    -   Eur. J. Pharmacol. (2010, 632, 93-102);    -   J. Chem. Inf. Model. (2011, 51, 115-129); and    -   Br. J. Pharmacol. (2015, 172, 3805-3816).

Nevertheless, there remains a need to identify and develop new kinaseinhibitors, specifically alternative p38 MAP kinase inhibitors that aresuitable for the treatment of inflammation. There is particularly a needfor such inhibitors that have improved therapeutic potential overcurrently available treatments or, in particular, that exhibit asuperior therapeutic index (e.g. inhibitors that are at least equallyefficacious and, in one or more respects, are less toxic at the relevanttherapeutic dose than previous agents).

SUMMARY OF THE INVENTION

We have now discovered, surprisingly, a benzoic acid aniline-possessingdiarylurea inhibits one or more of p38 MAP kinase, Syk and Src familykinases and therefore possess good anti-inflammatory properties.

Thus, according to a first aspect of the invention, there is provided acompound of formula I,

or a pharmaceutically acceptable salt thereof,which compound may be referred to hereinafter as “the compound of theinvention”.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Pharmaceutically acceptable salts that may be mentioned include acidaddition salts and base addition salts. Such salts may be formed byconventional means, for example by reaction of a free acid or a freebase form of a compound of formula I with one or more equivalents of anappropriate acid or base, optionally in a solvent, or in a medium inwhich the salt is insoluble, followed by removal of said solvent, orsaid medium, using standard techniques (e.g. in vacuo, by freeze-dryingor by filtration). Salts may also be prepared by exchanging acounter-ion of a compound of formula I in the form of a salt withanother counter-ion, for example using a suitable ion exchange resin.

Examples of pharmaceutically acceptable salts include acid additionsalts derived from mineral acids and organic acids, and salts derivedfrom metals.

For the avoidance of doubt, the compound of formula I may contain thestated atoms in any of their natural or non-natural isotopic forms. Inthis respect, embodiments of the invention that may be mentioned includethose in which:

-   (a) the compound of formula I is not isotopically enriched or    labelled with respect to any atoms of the compound; and-   (b) the compound of formula I is isotopically enriched or labelled    with respect to one or more atoms of the compound.

References herein to an “isotopic derivative” relate to the second ofthese two embodiments. In particular embodiments of the invention, thecompound of formula I is isotopically enriched or labelled (with respectto one or more atoms of the compound) with one or more stable isotopes.Thus, compounds of the invention that may be mentioned include, forexample, compounds of formula I that are isotopically enriched orlabelled with one or more atoms such as deuterium or the like.

The compound of formula I may exhibit tautomerism. All tautomeric formsand mixtures thereof are included within the scope of the invention.

Unless otherwise specified, alkyl groups and alkoxy groups as definedherein may be straight-chain or, when there is a sufficient number (i.e.a minimum of three) of carbon atoms, be branched. Particular alkylgroups that may be mentioned include, for example, methyl, ethyl,n-propyl, iso-propyl, butyl, n-butyl and tert-butyl. Particular alkoxygroups that may be mentioned include, for example, methoxy, ethoxy,propoxy, and butoxy.

Unless otherwise specified, alkylene groups as defined herein may bestraight-chain or, when there is a sufficient number (i.e. a minimum oftwo) of carbon atoms, be branched. In particular embodiments of theinvention, alkylene refers to straight-chain alkylene.

Unless otherwise stated, the point of attachment of aryl groups may bevia any atom of the ring system. However, when aryl groups are bicyclicor tricyclic, they are linked to the rest of the molecule via anaromatic ring. C₆₋₁₄ aryl groups include phenyl, naphthyl and the like.Embodiments of the invention that may be mentioned include those inwhich aryl is phenyl.

Unless otherwise specified, the term “halo” includes references tofluoro, chloro, bromo or iodo, in particular to fluoro, chloro or bromo,especially fluoro or chloro.

The compound of formula I has the name4-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-2-methoxybenzoicacid but may also be known by the chemical name4-[[4-[[4-[[5-tert-butyl-3-(methanesulfonamido)-2-methoxyphenyl]-carbamoylamino]-1-naphthyl]oxy]-2-pyridyl]amino]-2-methoxy-benzoicacid.

Examples of salts of the compound of formula I include allpharmaceutically acceptable salts, such as, without limitation, acidaddition salts of strong mineral acids such as HCl, H₂SO₄ and HBr salts(e.g. HCl or HBr salts) and addition salts of strong organic acids suchas methanesulfonic acid.

Particular salts of the compound of formula I that may be mentionedinclude hydrochloric acid salts and sodium, ammonium, calcium,magnesium, N-methylglucamine((2R,3R,4R,5S)-6-(methylamino)-hexane-1,2,3,4,5-pentol) or benethamine(N-benzyl-2-phenethylamine) salts (e.g. sodium or ammonium salts).

Thus, embodiments of the invention that may be mentioned include thehydrochloride, sodium, calcium, magnesium or ammonium salts of4-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-2-methoxybenzoicacid.

More particular salts of the compound of formula I that may be mentionedinclude the hydrochloride, sodium and ammonium salts.

References herein to a compound of the invention (a compound of formulaI) are intended to include references to the compound and to allpharmaceutically acceptable salts, solvates, isotopic derivatives and/ortautomers of said compound, unless the context specifically indicatesotherwise. In this respect, solvates that may be mentioned includehydrates.

The compound of the invention (compound of formula I) is a p38 MAPkinase inhibitor (especially of the alpha subtype), Syk kinase and Srcfamily kinases, e.g., Src and Lck, and is therefore useful in medicine,in particular for the treatment of inflammatory diseases. Furtheraspects of the invention that may be mentioned therefore include thefollowing.

-   -   (a) A pharmaceutical formulation comprising a compound of        formula I, as hereinbefore defined, or pharmaceutically        acceptable salt thereof, in admixture with a pharmaceutically        acceptable adjuvant, diluent or carrier.    -   (b) A combination product comprising        -   (A) a compound of formula I, as hereinbefore defined, or            pharmaceutically acceptable salt thereof, and        -   (B) another therapeutic agent,        -   wherein each of components (A) and (B) is formulated in            admixture with a pharmaceutically-acceptable adjuvant,            diluent or carrier.        -   In this aspect of the invention, the combination product may            be either a single (combination) pharmaceutical formulation            or a kit-of-parts.        -   Thus, this aspect of the invention encompasses a            pharmaceutical formulation including a compound of formula            I, as hereinbefore defined, or pharmaceutically acceptable            salt thereof, and another therapeutic agent, in admixture            with a pharmaceutically acceptable adjuvant, diluent or            carrier (which formulation is hereinafter referred to as a            “combined preparation”).        -   It also encompasses a kit of parts comprising components:        -   (i) a pharmaceutical formulation including a compound of            formula I, as hereinbefore defined, or pharmaceutically            acceptable salt thereof, in admixture with a            pharmaceutically acceptable adjuvant, diluent or carrier;            and        -   (ii) a pharmaceutical formulation including another            therapeutic agent, in admixture with a            pharmaceutically-acceptable adjuvant, diluent or carrier,        -   which components (i) and (ii) are each provided in a form            that is suitable for administration in conjunction with the            other.        -   Component (i) of the kit of parts is thus component (A)            above in admixture with a pharmaceutically acceptable            adjuvant, diluent or carrier. Similarly, component (ii) is            component (B) above in admixture with a pharmaceutically            acceptable adjuvant, diluent or carrier.    -   (c) A process for preparing the pharmaceutical formulation of        aspect (a) above, said process comprising the step of admixing        the compound of formula I, as hereinbefore defined, or        pharmaceutically acceptable salt thereof, with a        pharmaceutically acceptable adjuvant, diluent or carrier.        -   Embodiments of this aspect of the invention that may be            mentioned include those in which the pharmaceutically            acceptable adjuvant, diluent or carrier is a topically            acceptable adjuvant, diluent or carrier (and/or wherein the            process is for preparing a topical pharmaceutical            formulation, i.e. a pharmaceutical formulation that is            adapted for topical administration).    -   (d) A compound of formula I, as hereinbefore defined, or        pharmaceutically acceptable salt thereof, for use in medicine        (or for use as a medicament or as a pharmaceutical).    -   (e) A compound of formula I, as hereinbefore defined, or        pharmaceutically acceptable salt thereof, or a pharmaceutical        formulation or combination product as defined in connection with        aspect (a) or (b) of the invention, for use in the treatment or        prevention of an inflammatory disease.    -   (f) The use of        -   a compound of formula I, as hereinbefore defined, or            pharmaceutically acceptable salt thereof, or        -   a pharmaceutical formulation or combination product as            defined in connection with aspect (a) or (b) of the            invention,        -   for the preparation of a medicament for the treatment or            prevention of an inflammatory disease.    -   (g) A method of treating or preventing an inflammatory disease,        said method comprising administering to a subject an effective        amount of        -   a compound of formula I, as hereinbefore defined, or            pharmaceutically acceptable salt thereof, or        -   a pharmaceutical formulation or combination product as            defined in connection with aspect (a) or (b) of the            invention.    -   (h) A method of sensitizing a subject to the anti-inflammatory        effects of a corticosteroid, said method comprising        administering to the subject an effective amount of        -   a compound of formula I, as hereinbefore defined, or            pharmaceutically acceptable salt thereof, or        -   a pharmaceutical formulation or combination product as            defined in connection with aspect (a) or (b) of the            invention.        -   Embodiments of this aspect of the invention that may be            mentioned include those in which the subject is one who has            become refractory to the anti-inflammatory effects of a            corticosteroid.

References herein to “preventing an inflammatory disease” includereferences to preventing (or reducing the likelihood of) the recurrenceof an inflammatory disease in a subject who has previously suffered fromsuch a disease (e.g. a subject who has previously received treatment forthat disease, for example treatment according to the method described in(g) above).

Thus, still further aspects of the invention that may be mentionedinclude the following.

-   -   (i) A compound of formula I, as hereinbefore defined, or        pharmaceutically acceptable salt thereof, or a pharmaceutical        formulation or combination product as defined in connection with        aspect (a) or (b) of the invention, for use in reducing the        likelihood of the recurrence of an inflammatory disease in a        subject who has previously received treatment for that disease        (e.g. treatment with a compound of formula I, as hereinbefore        defined, or pharmaceutically acceptable salt thereof, or a        pharmaceutical formulation or combination product as defined in        connection with aspect (a) or (b) of the invention).    -   (j) The use of        -   a compound of formula I, as hereinbefore defined, or            pharmaceutically acceptable salt thereof, or        -   a pharmaceutical formulation or combination product as            defined in connection with aspect (a) or (b) of the            invention,        -   for the preparation of a medicament for reducing the            likelihood of the recurrence of an inflammatory disease in a            subject who has previously received treatment for that            disease (e.g. treatment with a compound of formula I, as            hereinbefore defined, or pharmaceutically acceptable salt            thereof, or a pharmaceutical formulation or combination            product as defined in connection with aspect (a) or (b) of            the invention).    -   (k) A method of reducing the likelihood of the recurrence of an        inflammatory disease in a subject who has previously received        treatment for that disease (e.g. treatment with a compound of        formula I, as hereinbefore defined, or pharmaceutically        acceptable salt thereof, or a pharmaceutical formulation or        combination product as defined in connection with aspect (a)        or (b) of the invention), said method comprising administering        to said subject an effective amount of        -   a compound of formula I, as hereinbefore defined, or            pharmaceutically acceptable salt thereof, or        -   a pharmaceutical formulation or combination product as            defined in connection with aspect (a) or (b) of the            invention.            Formulations

In relation to aspects (a) and (b) above, diluents and carriers that maybe mentioned include those suitable for parenteral, oral, topical,mucosal and rectal administration.

The pharmaceutical formulations and combination products of aspects (a)and (b) above may be prepared e.g. for parenteral, subcutaneous,intramuscular, intravenous, intra-articular, intravitreous, periocular,retrobulbar, subconjunctival, sub-Tenon, topical ocular orperi-articular administration, particularly in the form of liquidsolutions, emulsions or suspensions; for oral administration,particularly in the form of tablets or capsules, and especiallyinvolving technologies aimed at furnishing colon-targeted drug release(Patel, M. M. Expert Opin. Drug Deliv. 2011, 8 (10), 1247-1258); fortopical e.g. pulmonary or intranasal administration, particularly in theform of powders, nasal drops or aerosols and transdermal administration;for topical ocular administration, particularly in the form ofsolutions, emulsions, suspensions, ointments, implants/inserts, gels,jellies or liposomal microparticle formulations (Ghate, D.; Edelhauser,H. F. Expert Opin. Drug Deliv. 2006, 3 (2), 275-287); for ocularadministration, particularly in the form of biodegradable andnon-biodegradable implants, liposomes and nanoparticles (Thrimawithana,T. R. et al. Drug Discov. Today 2011, 16 (5/6), 270-277); for mucosaladministration e.g. to buccal, sublingual or vaginal mucosa, and forrectal administration e.g. in the form of a suppository or enema.

The pharmaceutical formulations and combination products of aspects (a)and (b) above may conveniently be administered in unit dosage form andmay be prepared by any of the methods well-known in the pharmaceuticalart, for example as described in Remington's Pharmaceutical Sciences,17th ed., Mack Publishing Company, Easton, Pa., (1985). Formulations forparenteral administration may contain as excipients sterile water orsaline, alkylene glycols such as propylene glycol, polyalkylene glycolssuch as polyethylene glycol, oils of vegetable origin, hydrogenatednaphthalenes and the like. Formulations for nasal administration may besolid and may contain excipients, for example, lactose or dextran, ormay be aqueous or oily solutions for use in the form of nasal drops ormetered sprays. For buccal administration, typical excipients includesugars, calcium stearate, magnesium stearate, pregelatinised starch, andthe like.

Pharmaceutical formulations and combination products suitable for oraladministration may comprise one or more physiologically compatiblecarriers and/or excipients and may be in solid or liquid form. Tabletsand capsules may be prepared with binding agents, for example, syrup,acacia, gelatin, sorbitol, tragacanth, or poly-vinylpyrrolidone;fillers, such as lactose, sucrose, corn starch, calcium phosphate,sorbitol, or glycine; lubricants, such as magnesium stearate, talc,polyethylene glycol, or silica; and surfactants, such as sodium laurylsulfate. Liquid compositions may contain conventional additives such assuspending agents, for example sorbitol syrup, methyl cellulose, sugarsyrup, gelatin, carboxymethyl-cellulose, or edible fats; emulsifyingagents such as lecithin, or acacia; vegetable oils such as almond oil,coconut oil, cod liver oil, or peanut oil; preservatives such asbutylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT).Liquid compositions may be encapsulated in, for example, gelatin toprovide a unit dosage form.

Solid oral dosage forms include tablets, two-piece hard shell capsulesand soft elastic gelatin (SEG) capsules. Such two-piece hard shellcapsules may be made from, for example, gelatin or hydroxylpropylmethylcellulose (HPMC).

A dry shell formulation typically comprises of about 40% to 60% w/wconcentration of gelatin, about a 20% to 30% concentration ofplasticizer (such as glycerin, sorbitol or propylene glycol) and about a30% to 40% concentration of water. Other materials such aspreservatives, dyes, opacifiers and flavours also may be present. Theliquid fill material comprises a solid drug that has been dissolved,solubilized or dispersed (with suspending agents such as beeswax,hydrogenated castor oil or polyethylene glycol 4000) or a liquid drug invehicles or combinations of vehicles such as mineral oil, vegetableoils, triglycerides, glycols, polyols and surface-active agents.

The compound of the invention may be administered topically (e.g. to thelung, eye or intestines). Thus, embodiments of aspects (a) and (b) abovethat may be mentioned include pharmaceutical formulations andcombination products that are adapted for topical administration. Suchformulations include those in which the excipients (including anyadjuvant, diluent and/or carrier) are topically acceptable.

Topical administration to the lung may be achieved by use of an aerosolformulation. Aerosol formulations typically comprise the activeingredient suspended or dissolved in a suitable aerosol propellant, suchas a chlorofluorocarbon (CFC) or a hydrofluorocarbon (HFC). Suitable CFCpropellants include trichloromonofluoromethane (propellant 11),dichlorotetrafluoroethane (propellant 114), and dichlorodifluoromethane(propellant 12). Suitable HFC propellants include tetrafluoroethane(HFC-134a) and heptafluoropropane (HFC-227). The propellant typicallycomprises 40% to 99.5% e.g. 40% to 90% by weight of the total inhalationcomposition. The formulation may comprise excipients includingco-solvents (e.g. ethanol) and surfactants (e.g. lecithin, sorbitantrioleate and the like). Other possible excipients include polyethyleneglycol, polyvinylpyrrolidone, glycerine and the like. Aerosolformulations are packaged in canisters and a suitable dose is deliveredby means of a metering valve (e.g. as supplied by Bespak, Valois or 3Mor alternatively by Aptar, Coster or Van).

Topical administration to the lung may also be achieved by use of anon-pressurised formulation such as an aqueous solution or suspension.This may be administered by means of a nebuliser e.g. one that can behand-held and portable or for home or hospital use (i.e. non-portable).The formulation may comprise excipients such as water, buffers, tonicityadjusting agents, pH adjusting agents, surfactants and co-solvents.Suspension liquid and aerosol formulations (whether pressurised orunpressurised) will typically contain the compound of the invention infinely divided form, for example with a D₅₀ of 0.5-10 μm e.g. around 1-5μm. Particle size distributions may be represented using D₁₀, D₅₀ andD₉₀ values. The D₅₀ median value of particle size distributions isdefined as the particle size in microns that divides the distribution inhalf. The measurement derived from laser diffraction is more accuratelydescribed as a volume distribution, and consequently the D₅₀ valueobtained using this procedure is more meaningfully referred to as a Dv₅₀value (median for a volume distribution). As used herein Dv values referto particle size distributions measured using laser diffraction.Similarly, D₁₀ and D₉₀ values, used in the context of laser diffraction,are taken to mean Dv₁₀ and Dv₉₀ values and refer to the particle sizewhereby 10% of the distribution lies below the D₁₀ value, and 90% of thedistribution lies below the D₉₀ value, respectively.

Topical administration to the lung may also be achieved by use of adry-powder formulation. A dry powder formulation will contain thecompound of the disclosure in finely divided form, typically with a massmean aerodynamic diameter (MMAD) of 1-10 μm or a D₅₀ of 0.5-10 μm e.g.around 1-5 μm. Powders of the compound of the invention in finelydivided form may be prepared by a micronization process or similar sizereduction process. Micronization may be performed using a jet mill suchas those manufactured by Hosokawa Alpine. The resultant particle sizedistribution may be measured using laser diffraction (e.g. with aMalvern Mastersizer 2000S instrument). The formulation will typicallycontain a topically acceptable diluent such as lactose, glucose ormannitol (preferably lactose), usually of large particle size e.g. anMMAD of 50 μm or more, e.g. 100 μm or more or a D₅₀ of 40-150 μm. Asused herein, the term “lactose” refers to a lactose-containingcomponent, including α-lactose monohydrate, β-lactose monohydrate,α-lactose anhydrous, β-lactose anhydrous and amorphous lactose. Lactosecomponents may be processed by micronization, sieving, milling,compression, agglomeration or spray drying. Commercially available formsof lactose in various forms are also encompassed, for example Lactohale®(inhalation grade lactose; DFE Pharma), InhaLac®70 (sieved lactose fordry powder inhaler; Meggle), Pharmatose® (DFE Pharma) and Respitose®(sieved inhalation grade lactose; DFE Pharma) products. In oneembodiment, the lactose component is selected from the group consistingof α-lactose monohydrate, α-lactose anhydrous and amorphous lactose.Preferably, the lactose is α-lactose monohydrate.

Dry powder formulations may also contain other excipients such as sodiumstearate, calcium stearate or magnesium stearate.

A dry powder formulation is typically delivered using a dry powderinhaler (DPI) device. Examples of dry powder delivery systems includeSPINHALER, DISKHALER, TURBOHALER, DISKUS and CLICKHALER. Furtherexamples of dry powder delivery systems include ECLIPSE, NEXT,ROTAHALER, HANDIHALER, AEROLISER, CYCLOHALER, BREEZHALER/NEOHALER,MONODOSE, FLOWCAPS, TWINCAPS, X-CAPS, TURBOSPIN, ELPENHALER, MIATHALER,TWISTHALER, NOVOLIZER, PRESSAIR, ELLIPTA, ORIEL dry powder inhaler,MICRODOSE, PULVINAL, EASYHALER, ULTRAHALER, TAIFUN, PULMOJET, OMNIHALER,GYROHALER, TAPER, CONIX, XCELOVAIR and PROHALER.

In one embodiment the compound of the present invention is provided in amicronized dry powder formulation, for example further comprisinglactose of a suitable grade optionally together with magnesium stearate,filled into a single dose device such as AEROLISER or filled into amulti dose device such as DISKUS.

The compound of the present invention may also be administered rectally,for example in the form of suppositories or enemas, which includeaqueous or oily solutions as well as suspensions and emulsions. Suchcompositions are prepared following standard procedures, well known bythose skilled in the art. For example, suppositories can be prepared bymixing the active ingredient with a conventional suppository base suchas cocoa butter or other glycerides, e.g. Suppocire. In this case, thedrug is mixed with a suitable non-irritating excipient which is solid atordinary temperatures but liquid at the rectal temperature and willtherefore melt in the rectum to release the drug. Such materials arecocoa butter and polyethylene glycols.

Generally, for compositions intended to be administered topically to theeye in the form of eye drops or eye ointments, the total amount of theinhibitor will be about 0.0001 to less than 4.0% (w/w).

Preferably, for topical ocular administration, the compositionsadministered according to the present invention will be formulated assolutions, suspensions, emulsions and other dosage forms. Aqueoussolutions are generally preferred, based on ease of formulation, as wellas a patient's ability to administer such compositions easily by meansof instilling one to two drops of the solutions in the affected eyes.However, the compositions may also be suspensions, viscous orsemi-viscous gels, or other types of solid or semi-solid compositions.Suspensions may be preferred for compounds that are sparingly soluble inwater.

The compositions administered according to the present invention mayalso include various other ingredients, including, but not limited to,tonicity agents, buffers, surfactants, stabilizing polymer,preservatives, co-solvents and viscosity building agents. Preferredpharmaceutical compositions of the present invention include theinhibitor with a tonicity agent and a buffer. The pharmaceuticalcompositions of the present invention may further optionally include asurfactant and/or a palliative agent and/or a stabilizing polymer.

Various tonicity agents may be employed to adjust the tonicity of thecomposition, preferably to that of natural tears for ophthalmiccompositions. For example, sodium chloride, potassium chloride,magnesium chloride, calcium chloride, simple sugars, such as dextrose,fructose, galactose, and/or simply polyols, such as the sugar alcoholsmannitol, sorbitol, xylitol, lactitol, isomaltitol, maltitol, andhydrogenated starch hydrolysates may be added to the composition toapproximate physiological tonicity. Such an amount of tonicity agentwill vary, depending on the particular agent to be added. In general,however, the compositions will have a tonicity agent in an amountsufficient to cause the final composition to have an ophthalmicallyacceptable osmolality (generally about 150-450 mOsm, preferably 250-350mOsm and most preferably at approximately 290 mOsm). In general, thetonicity agents of the invention will be present in the range of 2 to 5%w/w (e.g. 2 to 4% w/w). Preferred tonicity agents of the inventioninclude the simple sugars or the sugar alcohols, such as D-mannitol.

An appropriate buffer system (e.g. sodium phosphate, sodium acetate,sodium citrate, sodium borate or boric acid) may be added to thecompositions to prevent pH drift under storage conditions. Theparticular concentration will vary, depending on the agent employed.Preferably however, the buffer will be chosen to maintain a target pHwithin the range of pH 5 to 8, and more preferably to a target pH of pH5 to 7, or a target pH of 6.5 to 7.6.

Surfactants may optionally be employed to deliver higher concentrationsof inhibitor. The surfactants function to solubilise the inhibitor andstabilise colloid dispersion, such as micellar solution, microemulsion,emulsion and suspension. Examples of surfactants which may optionally beused include polysorbate, poloxamer, polyoxyl 40 stearate, polyoxylcastor oil, tyloxapol, triton, and sorbitan monolaurate. Preferredsurfactants to be employed in the invention have ahydrophile/lipophile/balance “HLB” in the range of 12.4 to 13.2 and areacceptable for ophthalmic use, such as TritonX114 and tyloxapol.

For example, a formulation of4-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)-phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-2-methoxybenzoicacid, or pharmaceutically acceptable salt thereof, for topical ocularadministration may comprise:

-   (a) water;-   (b) a surfactant (e.g. polyoxyl 40 stearate);-   (c) a tonicity agent (e.g. mannitol); and-   (d) an appropriate buffer system (e.g. a phosphate buffer containing    a mixture of monobasic dihydrogen phosphate and dibasic monohydrogen    phosphate) chosen to maintain a target pH within the range from 6.5    to 8.

In such topical ocular formulations, one or more (e.g. all) of thefollowing may apply:

-   (i)    4-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)-phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-2-methoxybenzoic    acid is present at a concentration in the range from 0.001 to 20    mg/mL (e.g. from 0.01 to 10 mg/mL, 0.1 to 2 mg/mL or, particularly,    1 mg/mL);-   (ii) the surfactant (e.g. polyoxyl 40 stearate) is present at from 1    to 10% w/w (e.g. from 2 to 5% w/w, such as from 2.5 to 4% w/w or,    particularly, 3% w/w);-   (iii) the tonicity agent (e.g. mannitol) is present at from 1 to 15%    w/w (e.g. from 2 to 10% w/w, such as from 3 to 6% w/w or,    particularly, 4.5% w/w);-   (iv) the buffer system used as a component of the formulation is an    aqueous phosphate buffer (e.g. a 10 mM aqueous phosphate buffer)    chosen to maintain a target pH within the range from 6.5 to 8.0    (e.g. within the range from 7.0 to 7.8 or, particularly, from 7.2 to    7.6).

Additional agents that may be added to the ophthalmic compositions ofthe present invention are demulcents which function as a stabilisingpolymer. The stabilizing polymer should be an ionic/charged example withprecedence for topical ocular use, more specifically, a polymer thatcarries negative charge on its surface that can exhibit a zeta-potentialof (−)10-50 mV for physical stability and capable of making a dispersionin water (i.e. water soluble). A preferred stabilising polymer of theinvention would be polyelectrolyte, or polyelectrolytes if more thanone, from the family of cross-linked polyacrylates, such as carbomers,polycarbophil and Pemulen(R), specifically Carbomer 974p (polyacrylicacid), at 0.1-0.5% w/w.

Other compounds may also be added to the ophthalmic compositions of thepresent invention to increase the viscosity of the carrier. Examples ofviscosity enhancing agents include, but are not limited to:polysaccharides, such as hyaluronic acid and its salts, chondroitinsulfate and its salts, dextrans, various polymers of the cellulosefamily, vinyl polymers and acrylic acid polymers.

Topical ophthalmic products are typically packaged in multidose form.Preservatives are thus required to prevent microbial contaminationduring use. Suitable preservatives include: benzalkonium chloride,chlorobutanol, benzododecinium bromide, methyl paraben, propyl paraben,phenylethyl alcohol, edentate disodium, sorbic acid, polyquaternium-1,or other agents known to those skilled in the art. Such preservativesare typically employed at a level of from 0.001 to 1.0% w/v. Unit dosecompositions of the present invention will be sterile, but typicallyunpreserved. Such compositions, therefore, generally will not containpreservatives.

The medical practitioner, or other skilled person, will be able todetermine a suitable dosage for the compound of the invention, and hencethe amount of the compound of the invention that should be included inany particular pharmaceutical formulation (whether in unit dosage formor otherwise).

Embodiments of the invention that may be mentioned in connection withthe combination products described at (b) above include those in whichthe other therapeutic agent is one or more therapeutic agents that areknown by those skilled in the art to be suitable for treatinginflammatory diseases (e.g. the specific diseases mentioned below).

For example, for the treatment of respiratory disorders (such as COPD orasthma), the other therapeutic agent is one or more agents selected fromthe list comprising:

-   -   steroids (e.g. budesonide, beclomethasone dipropionate,        fluticasone propionate, mometasone furoate, fluticasone furoate;        a further example is ciclesonide);    -   beta agonists, particularly beta2 agonists (e.g. terbutaline,        salbutamol, salmeterol, formoterol; further examples are        vilanterol, olodaterol, reproterol and fenoterol); and    -   xanthines (e.g. theophylline).

For example, for the treatment of respiratory disorders (such as COPD orasthma), the other therapeutic agent is one or more agents selected fromthe list comprising:

-   -   muscarinic antagonists (e.g. tiotropium, umeclidinium,        glycopyrronium, aclidinium and daratropium, any of these for        example as the bromide salt); and    -   phosphodiesterase inhibitors.

Further, for the treatment of gastrointestinal disorders (such asCrohn's disease or ulcerative colitis), the other therapeutic agent maybe, for example, one or more agents selected from the list comprising:

-   -   5-aminosalicylic acid, or a prodrug thereof (such as        sulfasalazine, olsalazine or balsalazide);    -   corticosteroids (e.g. prednisolone, methylprednisolone, or        budesonide);    -   immunosuppressants (e.g. cyclosporin, tacrolimus, methotrexate,        azathioprine or 6-mercaptopurine);    -   anti-TNFα antibodies (e.g. infliximab, adalimumab, certolizumab        pegol or golimumab);    -   anti-IL12/IL23 antibodies (e.g. ustekinumab) or small molecule        IL12/IL23 inhibitors (e.g. apilimod);    -   anti-α4β7 antibodies (e.g. vedolizumab);    -   toll-like receptor (TLR) blockers (e.g. BL-7040; Avecia        (Cambridge, UK));    -   MAdCAM-1 blockers (e.g. PF-00547659);    -   antibodies against the cell adhesion molecule α4-integrin (e.g.        natalizumab);    -   antibodies against the IL2 receptor a subunit (e.g. daclizumab        or basiliximab);    -   anti-Smad7 antibodies (e.g. mongersen (GED0301;        all-P-ambo-2′-deoxy-P-thioguanylyl-(3′→5′)-P-thiothymidylyl-(3′→5′)-2′-deoxy-5-methyl-P-thiocytidylyl-(3′→5′)-2′-deoxy-P-thioguanylyl-(3′→5′)-2′-deoxy-P-thiocytidylyl-(3′→5′)-2′-deoxy-P-thiocytidylyl-(3′→5′)-2′-deoxy-P-thiocytidylyl-(3′→5′)-2′-deoxy-P-thiocytidylyl-(3′→5′)-P-thiothymidylyl-(3′→5′)-P-thiothymidylyl-(3′→5′)-2′-deoxy-Pthiocytidylyl-(3′→5′)-P-thiothymidylyl-(3′→5′)-2′-deoxy-P-thiocytidylyl-(3′→5′)-2′-deoxy-P-thiocytidylyl-(3′→5′)-2′-deoxy-P-thiocytidylyl-(3′→5′)-2′-deoxy-5-methyl-P-thiocytidylyl-(3′→5′)-2′-deoxy-Pthioguanylyl-(3′→5′)-2′-deoxy-P-thiocytidylyl-(3′→5′)-2′-deoxy-Pthioadenylyl-(3′→5′)-2′-deoxy-P-thioguanylyl-(3′→5′)-2′-deoxycytidine));    -   sphingosine 1-phosphate receptor 1 (S1P1) modulators (e.g.        ozanimod        ((S)-5-(3-(1-((2-hydroxyethyl)amino)-2,3-dihydro-1H-inden-4-yl)-1,2,4-oxadiazol-5-yl)-2-isopropoxybenzonitrile),        amiselimod (MT1303;        2-amino-2-{2-[4-(heptyloxy)-3-(trifluoromethyl)phenyl]ethyl}propane-1,3-diol)        or APD334        (2-[7-[4-cyclopentyl-3-(trifluoromethyl)benzyloxy]-1,2,3,4-tetrahydrocyclopenta[b]indol-3(R)-yl]acetic        acid));    -   JAK inhibitors (e.g. tofacitinib, baricitinib        (1-(ethylsulfonyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]-3-azetidineacetonitrile),        filgotinib        (N-[5-[4-[(1,1-dioxo-1,4-thiazinan-4-yl)methyl]phenyl]-[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxamide),        peficitinib        (4-(((1R,2r,3S,5s,7s)-5-hydroxyadamantan-2-yl)amino)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide)        or R348 (see, for example, US 2014/0206708));    -   STAT3 inhibitors (e.g. TAK-114;        (3E)-1-methyl-3-(2-oxo-1H-indol-3-ylidene)indol-2-one);    -   receptor-interacting protein-1 (RIP1) kinase inhibitors (e.g.        GSK2982772);    -   Syk inhibitors and prodrugs thereof (e.g. fostamatinib and        R-406);    -   Phosphodiesterase-4 inhibitors (e.g. tetomilast);    -   HMPL-004;    -   probiotics;    -   microbiome modulators (e.g. SGM1019);    -   Dersalazine;    -   semapimod/CPSI-2364; and    -   protein kinase C inhibitors (e.g. AEB-071)

(e.g. for the treatment of gastrointestinal disorders (such as Crohn'sdisease or ulcerative colitis), the other therapeutic agent may be, forexample, one or more agents selected from the list comprising:

-   -   5-aminosalicylic acid, or a prodrug thereof (such as        sulfasalazine, olsalazine or balsalazide);    -   corticosteroids (e.g. prednisolone, methylprednisolone, or        budesonide);    -   immunosuppressants (e.g. cyclosporin, tacrolimus, methotrexate,        azathioprine or 6-mercaptopurine);    -   anti-TNFα antibodies (e.g. infliximab, adalimumab, certolizumab        pegol or golimumab);    -   anti-IL12/IL23 antibodies (e.g. ustekinumab) or small molecule        IL12/IL23 inhibitors (e.g. apilimod);    -   anti-α4β7 antibodies (e.g. vedolizumab);    -   MAdCAM-1 blockers (e.g. PF-00547659);    -   antibodies against the cell adhesion molecule α4-integrin (e.g.        natalizumab);    -   antibodies against the IL2 receptor α subunit (e.g. daclizumab        or basiliximab);    -   JAK3 inhibitors (e.g. tofacitinib or R348);    -   Syk inhibitors and prodrugs thereof (e.g. fostamatinib and        R-406);    -   Phosphodiesterase-4 inhibitors (e.g. tetomilast);    -   HMPL-004;    -   probiotics;    -   Dersalazine;    -   semapimod/CPSI-2364; and    -   protein kinase C inhibitors (e.g. AEB-071)).

For the treatment of eye disorders (such as uveitis andkeratoconjunctivitis sicca (dry eye)), the other therapeutic agent maybe, for example, one or more agents selected from the list comprising:

-   -   corticosteroids (e.g. dexamethasone, prednisolone, triamcinolone        acetonide, difluprednate or fluocinolone acetonide);    -   glucocorticoid agonists (e.g. mapracorat);    -   immunosuppressants (e.g. cyclosporin, voclosporin, azathioprine,        methotrexate, mycophenolate mofetil or tacrolimus);    -   anti-TNFα antibodies (e.g. infliximab, adalimumab, certolizumab        pegol, ESBA-105 or golimumab);    -   anti-IL-17A antibodies (e.g. secukinumab);    -   mTOR inhibitors (e.g. sirolimus);    -   VGX-1027;    -   adenosine A3 receptor agonists (e.g. CF-101);    -   lifitegrast;    -   IL1 blockers (e.g. EBI-005; Hou et al. PNAS 2013, 110(10),        3913-3918);    -   RGN-259 (Thymosin β4);    -   SI-614;    -   OTX-101;    -   JNK inhibitors (e.g. XG-104);    -   MAP kinase signalling inhibitors (e.g. DA-6034;        {[2-(3,4-dimethoxyphenyl)-5-methoxy-4-oxochromen-7-yl]oxy}acetic        acid);    -   mucin stimulators (e.g. rebamipide;        2-[(4-chlorobenzoyl)amino]-3-(2-oxo-1H-quinolin-4-yl)propanoic        acid);    -   MIM-D3 (Tavilermide; see, for example, US 2013/0345395);    -   JAK inhibitors (e.g. tofacitinib, baricitinib        (1-(ethylsulfonyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]-3-azetidineacetonitrile),        filgotinib        (N-[5-[4-[(1,1-dioxo-1,4-thiazinan-4-yl)methyl]phenyl]-[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxamide),        peficitinib        (4-(((1R,2r,3S,5s,7s)-5-hydroxyadamantan-2-yl)amino)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide)        or R348 (see, for example, US 2014/0206708)); and    -   protein kinase C inhibitors (e.g. AEB-071).

(e.g. for the treatment of eye disorders (such as uveitis andkeratoconjunctivitis sicca (dry eye)), the other therapeutic agent maybe, for example, one or more agents selected from the list comprising:

-   -   corticosteroids (e.g. dexamethasone, prednisolone, triamcinolone        acetonide, difluprednate or fluocinolone acetonide);    -   glucocorticoid agonists (e.g. mapracorat);—    -   immunosuppressants (e.g. cyclosporin, voclosporin, azathioprine,        methotrexate, mycophenolate mofetil or tacrolimus);    -   anti-TNFα antibodies (e.g. infliximab, adalimumab, certolizumab        pegol, ESBA-105 or golimumab);    -   anti-IL-17A antibodies (e.g. secukinumab);    -   mTOR inhibitors (e.g. sirolimus);    -   VGX-1027;    -   adenosine A3 receptor agonists (e.g. CF-101);    -   lifitegrast;    -   JAK3 inhibitors (e.g. tofacitinib or R348); and    -   protein kinase C inhibitors (e.g. AEB-071)).

In particular embodiments, for the treatment of eye disorders (such asuveitis and keratoconjunctivitis sicca (dry eye)), the other therapeuticagent may be, for example, one or more agents selected from the listcomprising:

-   -   corticosteroids (e.g. dexamethasone, prednisolone, triamcinolone        acetonide, difluprednate or fluocinolone acetonide);    -   immunosuppressants (e.g. cyclosporin, voclosporin, azathioprine,        methotrexate, mycophenolate mofetil or tacrolimus);    -   anti-TNFα antibodies (e.g. infliximab, adalimumab, certolizumab        pegol, ESBA-105 or golimumab);    -   anti-IL-17A antibodies (e.g. secukinumab);    -   mTOR inhibitors (e.g. sirolimus);    -   VGX-1027;    -   JAK inhibitors (e.g. tofacitinib, baricitinib, filgotinib,        peficitinib or R348) (e.g. JAK3 inhibitors such as tofacitinib        or R348); and    -   protein kinase C inhibitors (e.g. AEB-071).        Medical Uses

The compound of the invention may be used as monotherapies forinflammatory diseases, or in combination therapies for such diseases.

Thus, embodiments of aspects (e) to (g) above that may be mentionedinclude those in which the compound of formula I (or pharmaceuticallyacceptable salt thereof) is the sole pharmacologically active ingredientutilised in the treatment.

However, in other embodiments of aspects (e) to (g) above, the compoundof formula I (or pharmaceutically acceptable salt thereof) isadministered to a subject who is also administered one or more othertherapeutic agents (e.g. wherein the one or more other therapeuticagents are as defined above in connection with combination products).

When used herein, the term “inflammatory disease” specifically includesreferences to any one or more of the following:

-   (i) lung diseases or disorders having an inflammatory component,    such as cystic fibrosis, pulmonary hypertension, lung sarcoidosis,    idiopathic pulmonary fibrosis or, particularly, COPD (including    chronic bronchitis and emphysema), asthma or paediatric asthma;-   (ii) skin diseases or disorders having an inflammatory component,    such as atopic dermatitis, allergic dermatitis, contact dermatitis    or psoriasis;-   (iii) nasal diseases or disorders having an inflammatory component,    such as allergic rhinitis, rhinitis or sinusitis;-   (iv) eye diseases or disorders having an inflammatory component,    such as conjunctivitis, allergic conjunctivitis, glaucoma, diabetic    retinopathy, macular oedema (including diabetic macular oedema),    central retinal vein occlusion (CRVO), dry and/or wet age related    macular degeneration (AMD), post-operative cataract inflammation,    or, particularly, keratoconjunctivitis sicca (dry eye, also known as    xerophthalmia), uveitis (including posterior, anterior and pan    uveitis), corneal graft and limbal cell transplant rejection; and-   (v) gastrointestinal diseases or disorders having an inflammatory    component, such as gluten sensitive enteropathy (coeliac disease),    eosinophilic esophagitis, intestinal graft versus host disease or,    particularly, Crohn's disease or ulcerative colitis.

References herein to diseases having an inflammatory component includereferences to diseases that involve inflammation, whether or not thereare other (non-inflammatory) symptoms or consequences of the disease.

According to a further aspect of the invention there is provided aprocess for the preparation of the compound of formula I which processcomprises:

-   (a) reaction of a compound of formula II,

with a compound of formula III,

wherein one of Z¹ and Z² is a structural fragment of formula IV

and the other of Z¹ and Z² is a structural fragment of formula V

for example under conditions known to those skilled in the art, such asat a temperature from ambient (e.g. 15 to 30° C.) to about 110° C. inthe presence of a suitable organic solvent (e.g. a polar aprotic solventsuch as DMF, THF, 1,4-dioxane, or mixtures thereof);

-   (b) reaction of a compound of formula IIa,

wherein Z¹ is as defined above, with a suitable azide-forming agent(i.e. a suitable source of a leaving group and activated azide ion, suchas diphenyl phosphorazidate; see, for example, Tetrahedron 1974, 30,2151-2157) under conditions known to those skilled in the art, such asat sub-ambient to ambient temperature (e.g. from an initial temperatureof about −5 to 5° C. to ambient temperature post-reaction) in thepresence of an amine base (e.g. triethylamine or a sterically hinderedbase such as N,N-diisopropylethylamine) and a suitable organic solvent(e.g. a polar aprotic solvent such as DMF, THF, 1,4-dioxane, or mixturesthereof), which reaction is followed, without isolation, by thermalrearrangement (e.g. under heating) of the intermediate acyl azide (offormula Z¹—C(O)—N₃) e.g. at ambient temperature (such as from 15 to 30°C.) to provide, in situ, a compound of formula II, which compound isthen reacted with a compound of formula III, as defined above, toprovide the compound of formula I;

-   (c) reaction of a compound of formula IIb,

wherein LG¹ represents a suitable leaving group (e.g. imidazolyl,chloro, or aryloxy, such as phenoxy) and Z¹ is as defined above, with acompound of formula III, as defined above, for example under conditionsknown to those skilled in the art, such as at ambient temperature (e.g.from ambient to 80° C.), optionally in the presence of an amine base(e.g. triethylamine or a sterically hindered base likeN,N-diisopropylethylamine) and a suitable organic solvent (e.g. anaprotic solvent, such as dichloromethane or an ester such as isopropylacetate);

-   (d) reaction of a compound of formula VI,

wherein LG² represents a suitable leaving group (e.g. a halo group suchas chloro or bromo), with a compound of formula VII,

for example under conditions known to those skilled in the art (e.g. asdescribed in J. Am. Chem. Soc. 2011, 133, 15686-15696), such as atelevated temperature (e.g. from 50 to 110° C.) in the presence of asuitable organic solvent (e.g. a polar aprotic solvent such as DMF, THF,1,4-dioxane, or mixtures thereof) and, optionally, an acidic catalyst(e.g. a sulfonic acid such as para-toluenesulfonic acid);

-   (e) deprotection of a protected derivative of the compound of    formula I, under conditions known to those skilled in the art,    wherein the protected derivative bears a protecting group on an O-    or N-atom of the compound of formula I (and, for the avoidance of    doubt, a protected derivative of one compound of formula I may or    may not represent another compound of formula I).

Examples of protected derivatives of the compound of formula I includethose where:

-   -   an O-atom is protected with a benzyl group, which benzyl group        may be removed by hydrogenation, for example in the presence of        a palladium catalyst (such as Pd/C);    -   an O-atom of a carboxylic acid is protected with an alkyl group        (such as methyl, ethyl or tert-butyl), which alkyl group may be        removed by either basic hydrolysis (e.g. for methyl or ethyl        groups, by a hydrolysis reaction using an alkali metal hydroxide        such as sodium hydroxide) or acid hydrolysis (e.g. for a        tert-butyl group, by a hydrolysis reaction using an acid such as        trifluoroacetic acid).

Protected derivatives of the compound of formula I also includecompounds of formula VIIa,

wherein Q^(x) represents —C(O)OR^(4′) and R^(4′) represents a C₁₋₄ alkylgroup (e.g. a C₄ alkyl group or a C₁₋₃ alkyl group, such as methyl).

Compounds of formula II may be prepared according to or by analogy withmethods known to those skilled in the art, for example by reaction of acompound of formula IIa, as defined above, with an azide-forming agent,followed by rearrangement of the intermediate acyl azide (as describedat (b) above; see, for example, Tetrahedron 1974, 30, 2151-2157).

Compounds of formula IIb may be prepared reaction of a compound offormula VIII,

wherein LG¹ is as hereinbefore defined, with a compound of formula IX,

wherein Z¹ is as hereinbefore defined, for example under conditionsknown to those skilled in the art.

Amines of formula IX may be prepared from carboxylic acids of formulaIIa through the route described in (b) above, where the intermediateisocyanate II is hydrolysed with water to give a carbamic acid thatloses carbon dioxide to furnish IX. By the same token, the intermediateisocyanate II can be reacted with an alcohol, such as t-butanol, togenerate a protected version of IX.

The compound of formula III in which Z² represents a structural fragmentof formula V, or the compound of formula IX in which Z¹ represents astructural fragment of formula V, may be synthesised employing the routeoutlined in Scheme 1 (see, for example: WO 2003/072569; and WO2008/046216), wherein LG³ and LG⁴ represent leaving groups, e.g. halogenor methanesulfonyl, and FG represents a real or latent NH₂ group, i.e.,a group that is readily transformed into an NH₂ group, such as nitro ora protected variant NH-PG², where PG² is a typical protecting group(see, for example: Greene, T. W.; Wuts, P. G. M. Protective Groups inOrganic Synthesis; Wiley, 4th revised edition, 2006; ISBN-10:0471697540), e.g. a carbamate ester or carboxamide. The sequence startswith the base-mediated S_(N)Ar displacement of LG³ in XI by the aroxidesformed when X is treated with base to generate ethers XII. The remaininghalogen or methanesulfonyl substituent (LG⁴) of the ether XII is thendisplaced (i) by an amine of formula VII in a second S_(N)Ar reaction or(ii) via a Buchwald coupling (see, for example, WO 2009/017838) with anamine of formula VII to furnish the desired compound (when FG is NH₂),or XIII (when FG is nitro or NH-PG²). When FG is nitro in XIII, the NH₂group may be revealed by a reduction reaction, typically done throughhydrogenation employing a suitable catalyst, e.g. palladium on carbon,or employing dissolving metal conditions, such as with iron in glacialacetic acid. Alternatively, when FG is a protecting group, the NH₂ groupmay be revealed by a deprotection reaction. Although only depicted astaking place in the final step of the sequence, it should be noted thatthe unmasking of the latent NH₂ group represented by FG can take placeat any stage in the synthetic route shown in Scheme 1.

In a similar manner, amines of formula IX in which Z¹ represents astructural fragment of formula IV may be synthesised by conversion of alatent to a real NH₂ group in a compound of formula XIIIa,

wherein FG′ is as defined for FG above, except that it does notrepresent NH₂.

Compounds of formula VI may be synthesised by analogy with the compoundof formula I (see, for example, alternative processes (a) to (c) above).For example, compounds of formula VI can be prepared by reaction of acompound of formula IIx with a compound of formula IIIx, wherein thecompounds of formulae IIx and IIIx take the same definitions as thecompounds of formulae II and III, with the exception that one of Z¹ andZ² represents a structural fragment of formula IV, as hereinbeforedefined, and the other of Z¹ and Z² represents a structural fragment offormula Va,

Compounds of formula VIIa may be prepared by analogy with the proceduresdescribed herein for preparation of the compound of formula I (see, forexample, processes (a) to (d) and Scheme 1 above).

For example, the —CO₂H group may be replaced by Q^(x) in:

-   -   the structural fragment of formula V (to give a structural        fragment of formula Vp, and corresponding compounds of formulae        IIp, IIap, IIbp and IIIp, in which Z¹ and Z² are replaced by        Z^(1p) and Z^(2p), respectively, wherein one of Z^(1p) and        Z^(2p) is a structural fragment of formula IV, as defined above,        and the other of Z^(1p) and Z^(2p) is a structural fragment of        formula Vp); or    -   the compound of formula VII (to give a compound of formula        VIIp).

Alternatively, compounds of formula VIIa may be prepared by converting,in a compound of formula XIIIb

the group FG to NH₂, wherein FG and Q^(x) are as hereinbefore defined(e.g. by converting FG to NH₂ as described above in connection withScheme 1), followed by reaction with, for example, a compound of formulaIIb where Z¹ represents a structural fragment of formula IV.

It will be understood by persons skilled in the art that compoundsrepresented by formulae II, IIx and IIb are generally reactiveintermediates. These intermediates may be formed in situ and reacteddirectly, without isolation, with compounds of formula III to providethe compound of formula I. Furthermore, it will be understood by thoseskilled in the art that the use of appropriate protective groups may berequired during the processes described above for any of the groups Z¹and Z² which possess chemically-sensitive functional groups, forexample, a hydroxyl group or an amino function.

Many of the compounds illustrated in the Schemes are either commerciallyavailable, or can be obtained using the cited procedures, or can bereadily prepared by conventional methods by those skilled in the art.See for example Regan, J. et al.; J. Med. Chem. 2003, 46, 4676-4686, WO2000/043384, WO 2007/053346, WO 2007/087448, WO 2007/089512, WO2009/117080 and WO 2014/027209.

Novel intermediates as described herein form an aspect of the invention.In this respect, further aspects of the invention relate to:

-   (i) a compound of formula II, IIa or IIb as hereinbefore defined,    wherein Z¹ represents a structural fragment of formula V, or a salt    or protected derivative thereof;-   (ii) a compound of formula III as hereinbefore defined, wherein Z²    represents a structural fragment of formula V, or a salt or    protected derivative thereof;-   (iii) a compound of formula VIIa as hereinbefore defined, or a salt    or protected derivative thereof; and-   (v) a compound of formula XIII or XIIIb as hereinbefore defined, or    a salt or protected derivative thereof.

Protected derivatives of the compounds of formulae III, VII, XIII andXIIIb include those in which the essential NH₂ group (or NH₂ grouprepresented by FG) is protected. In this respect, such protectedderivatives include amides or, particularly, carbamates of thosecompounds. For example, those protected derivatives include compounds inwhich a H-atom of the NH₂ group is replaced by:

-   -   R′—C(O)—, wherein R′ is H, C₁₋₈ alkyl, phenyl or benzyl, which        latter two groups are optionally substituted by one or more        groups selected from halo, hydroxy, methyl and methoxy; or    -   R″—O—C(O)—, wherein R″ is tert-butyl, phenyl, benzyl or        fluorenyl, which latter three groups are optionally substituted        by one or more groups selected from halo, hydroxy, methyl and        methoxy.

Protected derivatives of the compounds of formulae II, IIa, IIb, III,VII and XIII in which R⁴ represents —CO₂H additionally (oralternatively) include those in which the carboxyl moiety is protected.In this respect, such protected derivatives also include esters (e.g.C₁₋₈ alkyl esters, such as ethyl or, particularly, methyl esters) ofsuch compounds.

Those skilled in the art will appreciate that compounds of formula IIIin which Z² represents a structural fragment of formula V may beprotected at the essential NH₂ group and/or, when R⁴ represents —CO₂H,at the carboxyl moiety. In this respect, for example, particularprotected derivatives of compounds of formula III in which Z² representsa structural fragment of formula V that may be mentioned include:

-   methyl    4-((4-((4-aminonaphthalen-1-yl)oxy)pyridin-2-yl)amino)-2-methoxybenzoate;    and-   methyl    4-((4-((4-((tert-butoxycarbonyl)amino)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-2-methoxybenzoate.

Both methyl4-((4-((4-aminonaphthalen-1-yl)oxy)pyridin-2-yl)amino)-2-methoxybenzoateand methyl4-((4-((4-((tert-butoxycarbonyl)amino)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-2-methoxybenzoateare also compounds of formula XIIIb (in which Q^(x) represents —CO₂CH₃and, respectively, FG represents NH₂ or NH-PG², in which PG² representstert-butyloxycarbonyl).

Alternative embodiments of the invention relate to a compound that iseither:

-   (i) a protected derivative of compound of formula III in which Z²    represents a structural fragment of formula V; or-   (ii) a compound of formula XIIIb, or a protected derivative thereof,

provided that said compound is not:

-   methyl    4-((4-((4-aminonaphthalen-1-yl)oxy)pyridin-2-yl)amino)-2-methoxybenzoate;    or-   methyl    4-((4-((4-((tert-butoxycarbonyl)amino)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-2-methoxybenzoate.

Still further embodiments of the invention relate to a compound offormula VIIa as hereinbefore defined, provided that said compoundeither:

-   (a) is; or-   (b) is not-   methyl    4-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-2-methoxybenzoate.

The aspects of the invention described herein (e.g. the above-mentionedcompounds, combinations, methods and uses) may have the advantage that,in the treatment of the conditions described herein, they may be moreconvenient for the physician and/or patient than, be more efficaciousthan, be less toxic than, have better selectivity over, have a broaderrange of activity than, be more potent than, produce fewer side effectsthan, have a better pharmacokinetic and/or pharmacodynamic profile than,have more suitable solid state morphology than, have better long termstability than, or may have other useful pharmacological propertiesover, similar compounds, combinations, methods (treatments) or usesknown in the prior art for use in the treatment of those conditions orotherwise.

The compound of the invention may additionally (or alternatively):

-   -   exhibit a long duration of action and/or persistence of action        (e.g. in comparison to other previously disclosed p38 MAP kinase        inhibitors such as, for example, BIRB796);    -   exhibit potent inhibition of Syk (e.g. they may have an IC₅₀        against Syk of 500 nM or less, such as 350 nM or less);    -   not strongly inhibit GSK 3α (e.g. they may have an IC₅₀ against        GSK 3α of 1,000 nM or greater; such as 1,500, 2,000, 3,000,        4,000, 5,000, 6,000, 7,000, 8,000, 9,000 or 10,000 nM or        greater);    -   target a smaller portion of the kinome, i.e., with improved        selectivity, as illustrated by lowered KinomeScan Selectivity        Scores;    -   maintain a relatively high local drug concentration between        doses (e.g. a high local concentration relative to other        previously disclosed p38 MAP kinase inhibitors such as, for        example, BIRB796);    -   exhibit properties that are particularly suited to topical/local        administration (e.g. following topical/local administration, the        generation of high target tissue concentrations but low plasma        concentrations of the compound of formula I and/or rapid        clearance of the compound of formula I from plasma, for example        as a result of high renal or hepatic extraction);    -   exhibit little or no β-catenin induction and/or inhibition of        mitosis in cells;    -   not produce increases in binucleated cells containing        micronuclei in the human lymphocyte in vitro micronucleus test;    -   exhibit little or no time-dependent inhibition of members of the        cytochrome P450 superfamily;    -   show improved chemical stability in the presence of water (e.g.        stability to hydrolysis in aqueous mixtures at elevated        temperatures) compared to previously disclosed p38 MAP kinase        inhibitors such as, for example, BIRB796;    -   following administration to a patient, give rise to metabolites        associated with little or no safety (e.g. toxicity) concerns;    -   display reduced ocular irritancy or toxicity in both preclinical        species and humans following topical administration;    -   exhibit good aqueous solubility and/or cellular permeability;    -   be more readily formulated in aqueous solution in the pH range        7-8 with lower quantities of solubilising excipients;    -   have a high degree of crystallinity; and/or    -   exhibit little or no hygroscopicity in the solid state.        Experimental Methods        General Procedures

All starting materials and solvents were obtained either from commercialsources or prepared according to the literature citation. Unlessotherwise stated all reactions were stirred. Organic solutions wereroutinely dried over anhydrous magnesium sulfate. Hydrogenations wereperformed on a Thales H-cube flow reactor under the conditions stated orunder a balloon of hydrogen. Microwave reactions were performed in a CEMDiscover and Smithcreator microwave reactor, heating to a constanttemperature using variable power microwave irradiation.

Normal phase column chromatography was routinely carried out on anautomated flash chromatography system such as CombiFlash Companion orCombiFlash RF system using pre-packed silica (230-400 mesh, 40-63 μm)cartridges. SOX was purchased from Supelco and treated with 1Mhydrochloric acid prior to use. Unless stated otherwise the reactionmixture to be purified was first diluted with MeOH and made acidic witha few drops of AcOH. This solution was loaded directly onto the SOX andwashed with MeOH. The desired material was then eluted by washing with1% NH₃ in MeOH.

Analytical Methods

Analytical HPLC was carried out using a Waters Xselect CSH 018, 2.5 μm,4.6×30 mm column eluting with a gradient of 0.1% Formic Acid in MeCN in0.1% aqueous Formic Acid or a Waters Xbridge BEH 018, 2.5 μm, 4.6×30 mmcolumn eluting with a gradient of MeCN in aqueous 10 mM AmmoniumBicarbonate. UV spectra of the eluted peaks were measured using either adiode array or variable wavelength detector on an Agilent 1100 system.

Analytical LCMS was carried out using a Waters Xselect CSH 018, 2.5 μm,4.6×30 mm column eluting with a gradient of 0.1% Formic Acid in MeCN in0.1% aqueous Formic Acid or a Waters Xbridge BEH 018, 2.5 μm, 4.6×30 mmcolumn eluting with a gradient of MeCN in aqueous 10 mM AmmoniumBicarbonate. UV and mass spectra of the eluted peaks were measured usinga variable wavelength detector on either an Agilent 1200 or an AgilentInfinity 1260 LCMS with 6120 single quadrupole mass spectrometer withpositive and negative ion electrospray.

Preparative HPLC was carried out using a Waters Xselect CSH 018, 5 μm,19×50 mm column using either a gradient of either 0.1% Formic Acid inMeCN in 0.1% aqueous Formic Acid or a gradient of MeCN in aqueous 10 mMAmmonium Bicarbonate or employing a Waters Xbridge BEH 018, 5 μm, 19×50mm column using a gradient of MeCN in aqueous 10 mM AmmoniumBicarbonate. Fractions were collected following detection by UV at asingle wavelength measured by a variable wavelength detector on a Gilson215 preparative HPLC or Varian PrepStar preparative HPLC or by mass andUV at a single wavelength measured by a ZQ single quadrupole massspectrometer, with positive and negative ion electrospray, and a dualwavelength detector on a Waters FractionLynx LCMS.

¹H NMR Spectroscopy:

¹H NMR spectra were acquired on a Bruker Avance III spectrometer at 400MHz. Either the central peaks of chloroform-d, dimethylsulfoxide-d₆ oran internal standard of tetramethylsilane were used as references.

PREPARATION OF THE COMPOUND OF THE INVENTION Example 14-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-2-methoxybenzoicacid

(i) tert-Butyl (4-((2-chloropyridin-4-yl)oxy)naphthalen-1-yl)carbamate

Method 1

A mixture of 4-((2-chloropyridin-4-yl)oxy)naphthalen-1-amine (see, forexample, Ito, K. et al., WO 2010/112936, 7 Oct. 2010; 1000 mg, 3.69mmol) and di-tert-butyl dicarbonate (750 mg, 3.44 mmol) in t-BuOH (10mL) was stirred at reflux for 18 h. The mixture was diluted with water(15 mL) and the solid collected by filtration. The solid was trituratedin diethyl ether to yield the sub-title compound (1002 mg) as a palegrey solid.

¹H NMR (DMSO-d6) 400 MHz, δ: 9.37 (s, 1H), 8.28 (d, 1H), 8.16 (d, 1H),8.82 (dd, 1H), 7.66 (d, 1H), 7.66-7.54 (m, 2H), 7.40 (d, 1H), 7.03 (d,1H), 6.91 (dd, 1H), 1.52 (s, 9H).

LCMS m/z 371 (M+H)⁺ (ES⁺); 369 (M−H)⁻ (ES⁻)

Method 2

2-Chloro-4-fluoropyridine (33 mL, 365 mmol) was added to a mixture oftert-butyl (4-hydroxynaphthalen-1-yl)carbamate (85 g, 328 mmol) andCs₂CO₃ (139 g, 426 mmol) in DMSO (600 mL) and stirred at rt for 24 h.Water (1 L) was added, the mixture stirred for 1 h, then the precipitatefiltered off. The reaction was repeated on a further 85 g scale ofnaphthol. The combined precipitates were washed with water (2 L), ether(4×400 mL) and dried under vacuum at 70° C. for 72 h to afford thesub-title compound (201.6 g) as a light grey solid.

¹H NMR (400 MHz, DMSO-d6) δ 9.38 (s, 1H), 8.28 (d, 1H), 8.16 (d, 1H),7.82 (d, 1H), 7.67-7.56 (m, 3H), 7.40 (d, 1H), 7.03 (d, 1H), 6.92 (dd,1H), 1.52 (s, 9H).

LCMS m/z 371 (M+H)⁺ (ES⁺); 369 (M−H)⁻ (ES⁻)

(ii) Methyl4-((4-((4-((tert-butoxycarbonyl)amino)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-2-methoxybenzoate

Method 1

A suspension of the product from step (i) above (2.0 g, 5.39 mmol),methyl 4-amino-2-methoxybenzoate (1.0 g, 5.52 mmol), BINAP (300 mg,0.482 mmol) and cesium carbonate (3.5 g, 10.74 mmol) in 1,4-dioxane (30mL) was degassed with nitrogen for 10 min. Pd₂dba₃ (200 mg, 0.218 mmol)was added and the mixture was heated to 90° C. overnight. The mixturewas diluted with diethyl ether (60 mL) and filtered. The filtrate wasthen washed with water (2×100 mL), and saturated brine (50 mL). Theorganic phase was dried (MgSO₄), filtered and concentrated under reducedpressure to yield the crude product as a red foam. The crude product waspurified by chromatography on the Companion (80 g column, 20-50% EtOAcin hexane) to afford the sub-title compound (2.34 g) as a yellow foam.

¹H NMR (400 MHz, DMSO-d6) δ: 9.38 (s, 1H), 9.36 (s, 1H), 8.18 (d, 1H),8.14 (d, 1H), 7.83 (d, 1H), 7.54-7.66 (m, 5H), 7.37 (d, 1H), 7.22 (dd,1H), 6.69 (dd, 1H), 6.15 (d, 1H), 3.74 (s, 3H), 3.71 (s, 3H), 1.53 (s,9H).

LCMS m/z 516 (M+H)⁺ (ES⁺)

Method 2

A mixture of methyl 4-amino-2-methoxybenzoate (10.8 g, 59.6 mmol), theproduct from step (i) above (20.09 g, 54.2 mmol) and potassium carbonate(15 g, 109 mmol) in DMF (300 mL) was degassed with N₂ for 10 min.BrettPhos G3 precatalyst (1 g, 1.103 mmol) was added and the mixtureheated at 85° C. for 3 h. The mixture was cooled then partitionedbetween DCM (500 mL) and water (800 mL). The organic layer was washedwith water (500 mL), dried (MgSO₄), filtered and evaporated underreduced pressure. The residue was triturated with ether, filtered anddried to afford the sub-title compound (21.7 g) as a grey solid.

¹H NMR (400 MHz, DMSO-d6) δ 9.38 (s, 1H), 9.36 (s, 1H), 8.18 (d, 1H),8.14 (d, 1H), 7.83 (d, 1H), 7.54-7.66 (m, 5H), 7.38 (d, 1H), 7.22 (dd,1H), 6.69 (dd, 1H), 6.14 (d, 1H), 3.74 (s, 3H), 3.71 (s, 3H), 1.53 (s,9H).

LCMS m/z 516 (M+H)⁺ (ES⁺)

(iii) Methyl4-((4-((4-aminonaphthalen-1-yl)oxy)pyridin-2-yl)amino)-2-methoxybenzoate

TFA (7 mL, 91 mmol) was added to a solution of the product from step(ii) above (2.34 g, 4.08 mmol) in DCM (50 mL) and the reaction stirredfor 2 h. The solvents were evaporated and the residue partitionedbetween sat NaHCO₃ soln. (100 mL) and DCM (60 mL). The organics wereseparated, dried (MgSO₄), filtered and the solvent evaporated to affordthe sub-title compound (1.5 g) as a pale brown foam.

LCMS m/z 416 (M+H)⁺ (ES⁺)

(iv) Phenyl(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)carbamate

Phenyl chloroformate (0.750 mL, 5.98 mmol) was added to a stirredsolution of N-(3-amino-5-(tert-butyl)-2-methoxyphenyl)methanesulfonamide(see, for example, Cirillo, P. F. et al., WO 2002/083628, 24 Oct. 2002;1.5 g, 5.51 mmol) and NaHCO₃ (1.0 g, 11.90 mmol) in THF (15 mL) and DCM(15 mL) and the mixture was stirred for 2 h. The mixture was washed withwater (20 mL) and the organic layer separated, dried (MgSO₄), filteredand evaporated to a brown foam which was stirred in cyclohexane (20 mL)to afford the sub-title compound (2.05 g) as a colourless solid.

LCMS m/z 393 (M+H)⁺ (ES⁺); 391 (M−H)⁻ (ES⁻)

(v) Methyl4-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)-naphthalen-1-yl)oxy)pyridin-2-yl)amino)-2-methoxybenzoate

Method 1

Triethylamine (20 μL, 0.143 mmol) was added to a solution of the productfrom step (iv) above (300 mg, 0.764 mmol) and the product from step (ii)above (300 mg, 0.722 mmol) in iPrOAc (15 mL) at 65° C. (blocktemperature) and the mixture stirred overnight. The reaction was cooledto rt and concentrated in vacuo affording a pale brown foam. The foamwas slurried in Et₂O (10 mL) for 2 h and the resulting solid collectedby filtration, washing with further portions of Et₂O, affording thesub-title compound (433 mg) as a pale pink solid.

LCMS m/z 358 (M+2H)²⁺ (ES⁺)

Method 2

Triethylamine (600 μL, 4.30 mmol) was added to a solution of the productfrom step (iv) above (9.0 g, 22.93 mmol) and the product from step (iii)above (9.0 g, 21.66 mmol) in iPrOAc (300 mL) at 65° C. (blocktemperature) and the mixture stirred for 24 h. The reaction was cooledto room temperature and concentrated in vacuo affording a brown foam.The crude product was purified by chromatography on the Companion (330 gcolumn, 1-5% MeOH in DCM) to afford the sub-title compound (13.2 g) as apale pink solid.

¹H NMR (400 MHz, DMSO-d6) δ: 9.40 (s, 1H), 9.35 (s, 1H), 916 (s, 1H),8.93 (s, 1H), 8.31 (d, 1H), 8.17-8.20 (m, 2H), 8.13 (d, 1H), 7.87 (d,1H), 7.69-7.73 (m, 1H), 7.60-7.63 (m, 2H), 7.53 (d, 1H), 7.41 (d, 1H),7.24 (dd, 1H), 7.03 (d, 1H), 6.69 (dd, 1H), 6.17 (d, 1H), 3.81 (s, 3H),3.74 (s, 3H), 3.71 (s, 3H), 3.10 (s, 3H), 1.27 (s, 9H).

LCMS m/z 714 (M+H)⁺ (ES⁺)

(vi)4-((4-((4-(3-(5-(tert-Butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-2-methoxybenzoicacid

Method 1

An aqueous solution of lithium hydroxide monohydrate (25 mg, 0.596 mmol)in water (3 mL) was added to a solution of the product from step (v)above (433 mg, 0.540 mmol) in THF (2 mL) and methanol (1 mL) and themixture was stirred at rt overnight. Lithium hydroxide monohydrate (25mg, 0.596 mmol) was added and stirring was continued for a further 3days. The mixture was concentrated under reduced pressure to remove THFand methanol then diluted with water (25 mL). A solution of citric acidmonohydrate (250 mg, 1.190 mmol) in water (5 mL) was added and theresulting precipitate was collected by filtration to yield the titlecompound (360 mg) as a pale pink solid.

¹H NMR (400 MHz, DMSO-d6) δ 11.96 (br s, 1H), 9.39 (s, 1H), 9.31 (s,1H), 9.14 (s, 1H), 8.91 (s, 1H), 8.30 (d, 1H), 8.19 (d, 1H), 8.18 (d,1H), 8.12 (d, 1H), 7.87 (d, 1H), 7.71 (ddd, 1H), 7.63 (d, 1H), 7.61(ddd, 1H), 7.51 (d, 1H), 7.40 (d, 1H), 7.22 (dd, 1H), 7.02 (d, 1H), 6.68(dd, 1H), 6.16 (d, 1H), 3.81 (s, 3H), 3.75 (s, 3H), 3.10 (s, 3H), 1.27(s, 9H). 90% purity

LCMS m/z 700 (M+H)⁺ (ES⁺)

Method 2

To a stirred solution of the product from step (v) above (33.4 g, 45.9mmol) in THF (300 mL) was added NaOH (6M aq.) (85.0 mL, 510 mmol). MeOH(60 mL) was added and stirring continued for 28 h. The reaction wasconcentrated in vacuo affording a yellow solid. The material wassuspended in water (200 mL) and acidified with 6M HCl (100 mL) causing awhite solid to precipitate. The solid was collected by filtration,washing with water. The resulting solid was dried on the frit for 1 hthen further dried at 40° C. under vacuum affording the title compoundas the hydrochloride salt as a white solid.

¹H NMR (of hydrochloride salt; 400 MHz, DMSO-d6) δ: 9.80 (s, 1H), 9.59(s, 1H), 9.15 (s, 1H), 9.02 (s, 1H), 8.37 (d, 1H), 8.13-8.18 (m, 3H),7.86 (d, 1H), 7.70-7.74 (m, 1H), 7.62-7.66 (m, 2H), 7.44 (d, 1H), 7.35(s, 1H), 7.10 (d, 1H), 7.03 (d, 1H), 6.78 (d, 1H), 6.26 (d, 1H), 3.81(s, 3H), 3.75 (s, 3H), 3.10 (s, 3H), 1.27 (s, 9H).

LCMS m/z 700 (M+H)⁺ (ES⁺)

The hydrochloride salt was loaded in 2.0 g batches, dissolved in THF,onto a pre-conditioned cartridge of SCX resin (20 g of resin,conditioned in MeCN). The resin was washed with MeCN then the productreleased in 1% NH₃ in MeOH. The NH₃ fractions were combined andconcentrated in vacuo, affording the title compound (30 g) as the freeacid as a pale pink solid.

¹H NMR (of free acid; 400 MHz, DMSO-d6) δ: 9.56 (s, 1H), 9.28 (s, 1H),9.00 (s, 1H), 8.34 (d, 1H), 8.16-8.17 (m, 2H), 8.11 (d, 1H), 7.86 (d,1H), 7.69-7.71 (m, 1H), 7.57-7.63 (m, 2H), 7.48 (d, 1H), 7.40 (d, 1H),7.21 (dd, 1H), 7.03 (d, 1H), 6.66 (dd, 1H), 6.16 (d, 1H), 3.81 (s, 3H),3.73 (s, 3H), 3.09 (s, 3H), 1.27 (s, 9H).

LCMS m/z 700 (M+H)⁺ (ES⁺)

The free acid (1.0 g, 1.386 mmol) was suspended in an aqueous solutionof NaOH (0.057 g, 1.414 mmol) in water (25 mL). MeOH (5 mL) was addedand the mixture stirred until homogeneity was achieved. The resultingsolution was diluted with MeOH (20 mL) and concentrated in vacuo,affording a pale grey solid. The material was suspended in MeCN (5 mL),to which water (0.5 mL) was added and the suspension stirred over theweekend. The suspension was filtered and the resulting solid washed withMeCN (2×3 mL) and dried under vacuum at 50° C., affording the titlecompound as the sodium salt (940 mg) as a white solid.

¹H NMR (of sodium salt; 400 MHz, DMSO-d6) δ: 9.68 (s, 1H), 9.07 (s, 1H),9.02 (s, 1H), 8.35 (d, 1H), 8.08-8.13 (m, 2H), 8.02 (d, 1H), 7.85 (d,1H), 7.64-7.68 (m, 1H), 7.57-7.61 (m, 1H), 7.37-7.43 (m, 2H), 7.30 (s,1H), 7.11 (dd, 1H), 7.03 (d, 1H), 6.61 (dd, 1H), 6.12 (d, 1H), 3.80 (s,3H), 3.65 (s, 3H), 2.96 (s, 3H), 1.25 (s, 9H).

LCMS m/z 700 (M+H)⁺ (ES⁺)

Biological Testing: Experimental Methods

Enzyme Binding Assays (Kinomescan)

Kinase enzyme binding activities of the compound disclosed herein may bedetermined using a proprietary assay which measures active site-directedcompetition binding to an immobilized ligand (Fabian, M. A. et al.,Nature Biotechnol, 2005, 23:329-336). These assays may be conducted byDiscoverX (formerly Ambit; San Diego, Calif.). The percentage inhibitionproduced by incubation with a test compound may be calculated relativeto the non-inhibited control.

Enzyme Inhibition Assays

The enzyme inhibitory activities of the compound disclosed herein aredetermined by FRET using synthetic peptides labelled with both donor andacceptor fluorophores (Z-LYTE, Invitrogen Ltd., Paisley, UK).

p38 MAPKα Enzyme Inhibition

The following two assay variants can be used for determination of p38MAPKα inhibition.

Method 1

The inhibitory activities of test compounds against the p38 MAPKαisoform (MAPK14: Invitrogen) are evaluated indirectly by determining thelevel of activation/phosphorylation of the down-stream molecule,MAPKAP-K2. The p38 MAPKα protein (80 ng/mL, 2.5 μL) is mixed with thetest compound (2.5 μL of either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL or 0.004μg/mL) for 2 hr at RT. The mix solution (2.5 μL) of the p38α inactivetarget MAPKAP-K2 (Invitrogen, 600 ng/mL) and FRET peptide (8 μM; aphosphorylation target for MAPKAP-K2) is then added, then the kinasereaction is initiated by adding ATP (40 μM, 2.5 μL). The mixture isincubated for 1 hr at RT. Development reagent (protease, 5 μL) is addedfor 1 hr prior to detection in a fluorescence microplate reader(Varioskan® Flash, ThermoFisher Scientific).

Method 2

This method follows the same steps as Method 1 above, but utilises ahigher concentration of the p38 MAPKα protein (2.5 μL of 200 ng/mLprotein instead of 2.5 μL of 80 ng/mL protein) for mixing with the testcompound (tested at either 1 μg/mL, 0.1 μg/mL, 0.01 μg/mL or 0.001μg/mL).

p38 MAPKγ Enzyme Inhibition

The inhibitory activities of the compound of the invention againstp38MAPKγ (MAPK12: Invitrogen), are evaluated in a similar fashion tothat described hereinabove. The enzyme (800 ng/mL, 2.5 μL) is incubatedwith the test compound (2.5 μL of either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL,or 0.004 μg/mL) for 2 hr at RT. The FRET peptides (8 μM, 2.5 μL), andappropriate ATP solution (2.5 μL, 400 μM) are then added to theenzymes/compound mixtures and the whole is incubated for 1 hr.Development reagent (protease, 5 μL) is added for 1 hr prior todetection in a fluorescence microplate reader (Varioskan® Flash, ThermoScientific).

c-Src and Syk Enzyme Inhibition

The inhibitory activities of the compound of the invention against c-Srcand Syk enzymes (Invitrogen), are evaluated in a similar fashion to thatdescribed hereinabove. The relevant enzyme (3000 ng/mL or 2000 ng/mLrespectively, 2.5 μL) is incubated with the test compound (either 1μg/mL, 0.1 μg/mL, 0.01 μg/mL, or 0.001 μg/mL, 2.5 μL each) for 2 hr atRT. The FRET peptides (8 μM, 2.5 μL), and appropriate ATP solutions (2.5μL, 800 μM for c-Src, and 60 μM ATP for Syk) are then added to theenzymes/compound mixtures and the mixture incubated for 1 hr.Development reagent (protease, 5 μL) is added for 1 hr prior todetection in a fluorescence microplate reader (Varioskan® Flash,ThermoFisher Scientific).

GSK 3α Enzyme Inhibition

The following two assay variants can be used for determination of GSK 3αinhibition.

Method 1

The inhibitory activities of the compound of the invention against theGSK 3α enzyme isoform (Invitrogen), are evaluated by determining thelevel of activation/phosphorylation of the target peptide. The GSK3-αprotein (500 ng/mL, 2.5 μL) is mixed with the test compound (2.5 μL ateither 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL, or 0.004 μg/mL) for 2 hr at RT.The FRET peptide (8 μM, 2.5 μL), which is a phosphorylation target forGSK3α, and ATP (40 μM, 2.5 μL) are then added to the enzyme/compoundmixture and the resulting mixture incubated for 1 hr. Developmentreagent (protease, 5 μL) is added for 1 hr prior to detection in afluorescence microplate reader (Varioskan® Flash, ThermoFisherScientific).

In all cases, the site-specific protease cleaves non-phosphorylatedpeptide only and eliminates the FRET signal. Phosphorylation levels ofeach reaction are calculated using the ratio of coumarin emission(donor) over fluorescein emission (acceptor), for which high ratiosindicate high phosphorylation and low ratios indicate lowphosphorylation levels. The percentage inhibition of each reaction iscalculated relative to non-inhibited control and the 50% inhibitoryconcentration (IC₅₀ value) is then calculated from theconcentration-response curve.

Method 2

This method follows the same steps as Method 1 above, but utilises ashorter period of mixing of the test compound (105 minutes instead of 2hours) with the GSK3-α protein. In addition, the concentrations of testcompound employed are either 10 μg/mL, 1 μg/mL, 0.1 μg/mL, or 0.01 μg/mL

Cellular Assays

The compound of the invention was studied using one or more of thefollowing assays.

(a) Inhibition of p38 MAPKα and Lck in Jurkat Cells

Jurkat T cells were cultured in starve medium (RPMI 1640+5% FBS) for 24h prior to the experiment. Cells were harvested and resuspended at10×10⁶ cells/mL in starve medium and then plated into round-bottomed 96well plates at 1×10⁶ cells/well. Serial dilutions of test compound wereadded (1% final DMSO concentration) for 2 h prior to stimulation.Following pre-incubation with compound, cells were stimulated with H₂O₂(0.05% final) for 5 min. The reaction was stopped by centrifugation at2000 rpm (3 min, 4° C.), then the supernatant was removed and 100 μL ofcold fix/perm solution (BD Fix/Perm kit #554714) added. Plates wereincubated for 20 min at 4° C. before centrifugation and washing withsupplied 1× wash medium (BD Fix/Perm kit #554714). Cells were stainedfor either phospho-p38α (T180/182), supplied by Cell SignallingTechnology (9211s), or phospho-Lck (Y394), supplied by R&D (MAB7500).Antibodies were diluted to 5 μg/mL (R&D) or 1:200 (Cell SignallingTechnology) in wash medium, before being incubated 1 h at 4° C. in thedark. Following 3 repeat washes with ice cold wash buffer, secondaryantibody (anti-rabbit-FITC #F1362 or anti-mouse-FITC #F2883, both fromSigma) was added at a dilution of 1:1000 and incubated for 1 h at 4° C.in the dark. Cells were washed 3× times in cold wash buffer then,following a final wash in cold PBS, were resuspended in 150 μL cold PBS.Cells were analysed by flow cytometry using BD Accuri C6.

(aa) LPS-induced TNFα/IL-8 Release in d-U937 Cells

U937 cells, a human monocytic cell line, are differentiated tomacrophage-type cells by incubation with phorbol myristate acetate (PMA;100 ng/mL) for 48 to 72 hr. Cells are pre-incubated with finalconcentrations of test compound for 2 hr and are then stimulated with0.1 μg/mL of LPS (from E. Coli: 0111:64, Sigma) for 4 hr. Thesupernatant is collected for determination of TNFα and IL-8concentrations by sandwich ELISA (Duo-set, R&D systems). The inhibitionof TNFα production is calculated as a percentage of that achieved by 10μg/mL of BIRB796 at each concentration of test compound by comparisonagainst vehicle control. The relative 50% effective concentration(REC₅₀) is determined from the resultant concentration-response curve.The inhibition of IL-8 production is calculated at each concentration oftest compound by comparison with vehicle control. The 50% inhibitoryconcentration (IC₅₀) is determined from the resultantconcentration-response curve.

(b) LPS-induced TNFα/IL-8 Release in PBMC Cells

Peripheral blood mononuclear cells (PBMCs) from healthy subjects areseparated from whole blood using a density gradient (Lymphoprep,Axis-Shield Healthcare). The PBMCs are seeded in 96 well plates andtreated with compounds at the desired concentration for 2 hours beforeaddition of 1 ng/mL LPS (Escherichia Coli 0111:64 from Sigma Aldrich)for 24 hours under normal tissue culture conditions (37° C., 5% CO₂).The supernatant is harvested for determination of IL-8 and TNFαconcentrations by sandwich ELISA (Duo-set, R&D systems) and read on thefluorescence microplate reader (Varioskan® Flash, ThermoFisherScientific). The concentration at 50% inhibition (IC₅₀) of IL-8 and TNFαproduction is calculated from the dose response curve.

(c) IL-2 and IFN Gamma Release in CD3/CD28 Stimulated PBMC Cells

PBMCs from healthy subjects are separated from whole blood using adensity gradient (Lymphoprep, Axis-Shield Healthcare). Cells are addedto a 96 well plate pre-coated with a mixture of CD3/CD28 monoclonalantibodies (0.3 μg/mL eBioscience and 3 μg/mL BD Pharmingenrespectively). Compound at the desired concentration is then added tothe wells and the plate left for 3 days under normal tissue cultureconditions. Supernatants are harvested and IL-2 and IFN gamma releasedetermined by Sandwich ELISA (Duo-set, R&D System). The IC₅₀ isdetermined from the dose response curve.

(d) IL-1β-induced IL-8 Release in HT29 Cells

H129 cells, a human colon adenocarcinoma cell line, are plated in a 96well plate (24 hr) and pre-treated with compounds at the desiredconcentration for 2 hours before addition of 5 ng/mL of IL-1β (Abcam)for 24 hours. Supernatants are harvested for IL-8 quantification bySandwich ELISA (Duo-set, R&D System). The IC₅₀ is determined from thedose response curve.

(e) LPS-induced IL-8 and TNFα Release in Primary Macrophages

PBMCs from healthy subjects are separated from whole blood using adensity gradient (Lymphoprep, Axis-Shield Healthcare). Cells areincubated for 2 hrs and non-adherent cells removed by washing. Todifferentiate the cells to macrophages, they are incubated with 5 ng/mLof GM-CSF (Peprotech) for 7 days under normal tissue culture conditions.Compounds are then added to the cells at the desired concentration for a2 hour pre-treatment before stimulation with 10 ng/mL LPS for 24 hours.Supernatants are harvested and IL-8 and TNFα release determined bySandwich ELISA (Duo-set, R&D System). The IC₅₀ is determined from thedose response curve.

(f) Poly I:C-induced ICAM-1 Expression in BEAS2B Cells

Poly I:C is used in these studies as a simple, RNA virus mimic. PolyI:C-Oligofectamine mixture (1 μg/mL Poly I:C, ±2% Oligofectamine, 25 μL;Invivogen Ltd., San Diego, Calif., and Invitrogen, Carlsbad, Calif.,respectively) is transfected into BEAS2B cells (human bronchialepithelial cells, ATCC). Cells are pre-incubated with finalconcentrations of test compounds for 2 hr and the level of ICAM1expression on the cell surface is determined by cell-based ELISA. At atime point 18 hr after poly I:C transfection, cells are fixed with 4%formaldehyde in PBS and then endogenous peroxidase is quenched by theaddition of washing buffer (100 μL, 0.05% Tween in PBS: PBS-Tween)containing 0.1% sodium azide and 1% hydrogen peroxide. Cells are washedwith wash-buffer (3×200 μL) and after blocking the wells with 5% milk inPBS-Tween (100 μL) for 1 hr, the cells are incubated with anti-humanICAM-1 antibody (50 μL; Cell Signalling Technology, Danvers, Mass.) in1% BSA PBS overnight at 4° C.

The cells are washed with PBS-Tween (3×200 μL) and incubated with thesecondary antibody (100 μL; HRP-conjugated anti-rabbit IgG, Dako Ltd.,Glostrup, Denmark). The cells are then incubated with substrate (50 μL)for 2-20 min, followed by the addition of stop solution (50 μL, 1NH₂SO₄). The ICAM-1 signal is detected by reading the absorbance at 450nm against a reference wavelength of 655 nm using a spectrophotometer.The cells are then washed with PBS-Tween (3×200 μL) and total cellnumbers in each well are determined by reading absorbance at 595 nmafter Crystal Violet staining (50 μL of a 2% solution in PBS) andelution by 1% SDS solution (100 μL) in distilled water. The measured OD450-655 readings are corrected for cell number by dividing with theOD595 reading in each well. The inhibition of ICAM-1 expression iscalculated at each concentration of test compound by comparison withvehicle control. The 50% inhibitory concentration (IC₅₀) is determinedfrom the resultant concentration-response curve.

(g) Cell Mitosis Assay

Peripheral blood mononucleocytes (PBMCs) from healthy subjects areseparated from whole blood (Quintiles, London, UK) using a densitygradient (Histopaque®-1077, Sigma-Aldrich, Poole, UK). The PBMCs (3million cells per sample) are subsequently treated with 2% PHA(phytohaemagglutinin, Sigma-Aldrich, Poole, UK) for 48 hr, followed by a20 hr exposure to varying concentrations of test compounds. At 2 hrbefore collection, PBMCs are treated with demecolcine (0.1 μg/mL;Invitrogen, Paisley, UK) to arrest cells in metaphase. To observemitotic cells, PBMCs are permeabilised and fixed by adding Intraprep (50μL; Beckman Coulter, France), and stained with anti-phospho-histone 3(0.26 ng/L; #9701; Cell Signalling, Danvers, Mass.) and propidium iodide(1 mg/mL; Sigma-Aldrich, Poole, UK) as previously described (MuehlbauerP. A. and Schuler M. J., Mutation Research, 2003, 537:117-130).Fluorescence is observed using an ATTUNE flow cytometer (Invitrogen,Paisley, UK), gating for lymphocytes. The percentage inhibition ofmitosis is calculated for each treatment relative to vehicle (0.5% DMSO)treatment.

(h) Rhinovirus-induced IL-8 Release and ICAM-1 Expression

Human rhinovirus RV16 is obtained from the American Type CultureCollection (Manassas, Va.). Viral stocks are generated by infecting HeLacells with HRV until 80% of the cells are cytopathic.

BEAS2B cells are infected with HRV at an MOI of 5 and incubated for 2 hrat 33° C. with gentle shaking to promote absorption. The cells are thenwashed with PBS, fresh media added and the cells are incubated for afurther 72 hr. The supernatant is collected for assay of IL-8concentrations using a Duoset ELISA development kit (R&D systems,Minneapolis, Minn.).

The level of ICAM-1 expressing cell surface is determined by cell-basedELISA. At 72 hr after infection, cells are fixed with 4% formaldehyde inPBS. After quenching endogenous peroxidase by adding 0.1% sodium azideand 1% hydrogen peroxide, wells are washed with wash-buffer (0.05% Tweenin PBS: PBS-Tween). After blocking well with 5% milk in PBS-Tween for 1hr, the cells are incubated with anti-human ICAM-1 antibody in 5% BSAPBS-Tween (1:500) overnight. Wells are washed with PBS-Tween andincubated with the secondary antibody (HRP-conjugated anti-rabbit IgG,Dako Ltd.). The ICAM-1 signal is detected by adding substrate andreading at 450 nm with a reference wavelength of 655 nm using aspectrophotometer. The wells are then washed with PBS-Tween and totalcell numbers in each well are determined by reading absorbance at 595 nmafter Crystal Violet staining and elution with 1% SDS solution. Themeasured OD₄₅₀₋₆₅₅ readings are corrected for cell number by dividingwith the OD₅₉₅ reading in each well. Compounds are added 2 hr before HRVinfection and 2 hr after infection when non-infected HRV is washed out.

(i) Assessment of HRV16 Induced Cytopathic Effect (CPE) in MRC5 Cells

MRC5 cells are infected with HRV16 at an MOI of 1 in DMEM containing 5%FCS and 1.5 mM MgCl₂, followed by incubation for 1 hr at 33° C. topromote adsorption. The supernatants are aspirated, and then fresh mediaadded followed by incubation for 4 days. Where appropriate, cells arepre-incubated with compound or DMSO for 2 hr, and the compounds and DMSOadded again after washout of the virus.

Supernatants are aspirated and incubated with methylene blue solution(100 μL, 2% formaldehyde, 10% methanol and 0.175% Methylene Blue) for 2hr at RT. After washing, 1% SDS in distilled water (100 μL) is added toeach well, and the plates are shaken lightly for 1-2 hr prior to readingthe absorbance at 660 nm. The percentage inhibition for each well iscalculated. The IC₅₀ value is calculated from the concentration-responsecurve generated by the serial dilutions of the test compounds.

(j) In Vitro RSV Virus Load in Primary Bronchial Epithelial Cells

Normal human bronchial epithelial cells (NHBEC) grown in 96 well platesare infected with RSV A2 (Strain A2, HPA, Salisbury, UK) at a MOI of0.001 in the LHC8 Media:RPMI-1640 (50:50) containing 15 mM magnesiumchloride and incubated for 1 hr at 37° C. for adsorption. The cells arewashed with PBS (3×200 μL), then fresh media (200 μL) is added andincubation continued for 4 days. Where appropriate, cells arepre-incubated with the compound or DMSO for 2 hr, and then added againafter washout of the virus.

The cells are fixed with 4% formaldehyde in PBS solution (50 μL) for 20min, washed with WB (3×200 μL) (washing buffer, PBS including 0.5% BSAand 0.05% Tween-20) and incubated with blocking solution (5% condensedmilk in PBS) for 1 hr. Cells are then washed with WB (3×200 μL) andincubated for 1 hr at RT with anti-RSV (2F7) F-fusion protein antibody(40 μL; mouse monoclonal, lot 798760, Cat. No. ab43812, Abcam) in 5% BSAin PBS-tween. After washing, cells are incubated with an HRP-conjugatedsecondary antibody solution (50 μL) in 5% BSA in PBS-Tween (lot00053170, Cat. No. P0447, Dako) and then TMB substrate added (50 μL;substrate reagent pack, lot 269472, Cat. No. DY999, R&D Systems, Inc.).This reaction is stopped by the addition of 2N H₂SO₄ (50 μL) and theresultant signal is determined colourimetrically (OD: 450 nm with areference wavelength of 655 nm) in a microplate reader (Varioskan®Flash, ThermoFisher Scientific).

Cells are then washed and a 2.5% crystal violet solution (50 μL; lot8656, Cat. No. PL7000, Pro-Lab Diagnostics) is applied for 30 min. Afterwashing with WB, 1% SDS in distilled water (100 μL) is added to eachwell, and plates are shaken lightly on the shaker for 1 hr prior toreading the absorbance at 595 nm. The measured OD₄₅₀₋₆₅₅ readings arecorrected to the cell number by dividing the OD₄₅₀₋₆₅₅ by the OD₅₉₅readings. The percentage inhibition for each well is calculated and theIC₅₀ value is calculated from the concentration-response curve generatedfrom the serial dilutions of compound.

(k) Cell Viability Assay: MTT Assay

Differentiated U937 cells are pre-incubated with each test compound(final concentration 1 μg/mL or 10 μg/mL in 200 μL media indicatedbelow) under two protocols: the first for 4 hr in 5% FCS RPMI1640 mediaand the second in 10% FCS RPMI1640 media for 24 h. The supernatant isreplaced with new media (200 μL) and MTT stock solution (10 μL, 5 mg/mL)is added to each well. After incubation for 1 hr the media are removed,DMSO (200 μL) is added to each well and the plates are shaken lightlyfor 1 hr prior to reading the absorbance at 550 nm. The percentage lossof cell viability is calculated for each well relative to vehicle (0.5%DMSO) treatment. Consequently an apparent increase in cell viability fordrug treatment relative to vehicle is tabulated as a negativepercentage.

(l) Human Biopsy Assay

Intestinal mucosa biopsies are obtained from the inflamed regions of thecolons of IBD patients. The biopsy material is cut into small pieces(2-3 mm) and placed on steel grids in an organ culture chamber at 37° C.in a 5% CO₂/95% O₂ atmosphere in serum-free media. DMSO control or testcompounds at the desired concentration are added to the tissue andincubated for 24 hr in the organ culture chamber. The supernatant isharvested for determination of IL-6, IL-8, IL-1β and TNFα levels by R&DELISA. Percentage inhibition of cytokine release by the test compoundsis calculated relative to the cytokine release determined for the DMSOcontrol (100%).

(m) Accumulation of β Catenin in d-U937 Cells

U937 cells, a human monocytic cell line, are differentiated intomacrophage-type cells by incubation with PMA (100 ng/mL) for between 48to 72 hr. The cells are then incubated with either final concentrationsof test compound or vehicle for 18 hr. The induction of β-catenin by thetest compounds is stopped by replacing the media with 4% formaldehydesolution. Endogenous peroxide activity is neutralised by incubating withquenching buffer (100 μL, 0.1% sodium azide, 1% H₂O₂ in PBS with 0.05%Tween-20) for 20 min. The cells are washed with washing buffer (200 μL;PBS containing 0.05% Tween-20) and incubated with blocking solution (200μL; 5% milk in PBS) for 1 hr, re-washed with washing buffer (200 μL) andthen incubated overnight with anti-β-catenin antibody solution (50 μL)in 1% BSA/PBS (BD, Oxford, UK).

After washing with washing buffer (3×200 μL; PBS containing 0.05%Tween-20), cells are incubated with a HRP-conjugated secondary antibodysolution (100 μL) in 1% BSA/PBS (Dako, Cambridge, UK) and the resultantsignal is determined colourimetrically (OD: 450 nm with a referencewavelength of 655 nm) using TMB substrate (50 μL; R&D Systems, Abingdon,UK). This reaction is stopped by addition of 1N H₂SO₄ solution (50 μL).Cells are then washed with washing buffer and 2% crystal violet solution(50 μL) is applied for 30 min. After washing with washing buffer (3×200μL), 1% SDS (100 μL) is added to each well and the plates are shakenlightly for 1 hr prior to measuring the absorbance at 595 nm (Varioskan®Flash, Thermo-Fisher Scientific).

The measured OD₄₅₀₋₆₅₅ readings are corrected for cell number bydividing the OD₄₅₀₋₆₅₅ by the OD₅₉₅ readings. The percentage inductionfor each well is calculated relative to vehicle, and the ratio ofinduction normalised in comparison with the induction produced by astandard control comprising the Reference compoundN-(4-(4-(3-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)ureido)naphthalen-1-yloxy)pyridin-2-yl)-2-methoxyacetamide(1 μg/mL), which is defined as unity.

(n) T Cell Proliferation

PBMCs from healthy subjects are separated from whole blood using adensity gradient (Lymphoprep, Axis-Shield Healthcare). The lymphocytefraction is first enriched for CD4+ T cells by negative magnetic cellsorting as per the manufacturer's instructions (Miltenyi Biotec130-091-155). Naïve CD4+ T cells are then separated using positivemagnetic selection of CD45RA+ cells using microbeads as per themanufacturer's instructions (130-045-901). Cells are plated at 2×10⁵cells per well in 100 μL RPMI/10% FBS on 96 well flat bottomed plate(Corning Costar). 25 μL of test compound are diluted to the appropriateconcentration (8× final concentration) in normal medium and added toduplicate wells on the plate to achieve a dose response range of 0.03ng/mL-250 ng/mL. DMSO is added as a negative control. Plates are allowedto pre-incubate for 2 hours before stimulation with 1 μg/mL anti-CD3(OKT3; eBioscience). After 72 h, the medium in each well is replacedwith 150 μL of fresh medium containing 10 μM BrdU (Roche). After 16 h,the supernatant is removed, the plate is dried and the cells fixed byadding 100 μL of fix/denature solution to each well for 20 min as perthe manufacturer's instructions (Roche). Plates are washed once with PBSbefore addition of the anti-BrdU detection antibody and incubated for 90mins at room temperature. Plates are then washed gently 3× with the washbuffer supplied and developed by addition of 100 μL of substratesolution. The reaction is stopped by addition of 50 μL of 1 M H₂SO₄ andread for absorbance at 450 nm on a plate reader (Varioskan® Flash,ThermoFisher Scientific). The IC₅₀ is determined from the dose responsecurve.

(o) IL-2 and IFNγ Release in CD3/CD28 Stimulated LPMC Cells from IBDPatients

Lamina propria mononuclear cells (LPMCs) are isolated and purified frominflamed IBD mucosa of surgical specimens or from normal mucosa ofsurgical specimens as follows: The mucosa is removed from the deeperlayers of the surgical specimens with a scalpel and cut in fragments ofsize 3-4 mm. The epithelium is removed by washing the tissue fragmentsthree times with 1 mM EDTA (Sigma-Aldrich, Poole, UK) in HBSS(Sigma-Aldrich) with agitation using a magnetic stirrer, discarding thesupernatant after each wash. The sample is subsequently treated withtype 1A collagenase (1 mg/mL; Sigma-Aldrich) for 1 h with stirring at37° C. The resulting cell suspension is then filtered using a 100 μmcell strainer, washed twice, resuspended in RPMI-1640 medium(Sigma-Aldrich) containing 10% fetal calf serum, 100 U/mL penicillin and100 μg/mL streptomycin, and used for cell culture.

Freshly isolated LPMCs (2×10⁶ cells/well) are stimulated with 1 μg/mLα-CD3/α-CD28 for 48 h in the presence of either DMSO control orappropriate concentrations of compound. After 48 h, the supernatant isremoved and assayed for the presence of TNFα and IFNγ by R&D ELISA.Percentage inhibition of cytokine release by the test compounds iscalculated relative to the cytokine release determined for the DMSOcontrol (100%).

(p) Inhibition of Cytokine Release from Myofibroblasts Isolated from IBDPatients

Myofibroblasts from inflamed IBD mucosa are isolated as follows:

The mucosa is dissected and discarded and 1 mm-sized mucosal samples arecultured at 37° C. in a humidified CO₂ incubator in Dulbecco's modifiedEagle's medium (DMEM, Sigma-Aldrich) supplemented with 20% FBS, 1%non-essential amino acids (Invitrogen, Paisley, UK), 100 U/mLpenicillin, 100 μg/mL streptomycin, 50 μg/mL gentamycin, and 1 μg/mLamphotericin (Sigma-Aldrich). Established colonies of myofibroblasts areseeded into 25-cm² culture flasks and cultured in DMEM supplemented with20% FBS and antibiotics to at least passage 4 to provide a sufficientquantity for use in stimulation experiments.

Subconfluent monolayers of myofibroblasts, seeded in 12-well plates at3×10⁶ cells per well, are starved in serum-free medium for 24 h at 37°C., 5% CO₂, before being cultured for 24 h in the presence of eitherDMSO control or appropriate concentrations of compound. After 24 h, thesupernatant is removed and assayed for the presence of IL-8 and IL-6 byR&D ELISA. Percentage inhibition of cytokine release by the testcompounds is calculated relative to the cytokine release determined forthe DMSO control (100%).

(q) Human Neutrophil Degranulation

Neutrophils are isolated from human peripheral blood as follows:

Blood is collected by venepuncture and anti-coagulated by addition of1:1 EDTA:sterile phosphate buffered saline (PBS, no Ca+/Mg+). Dextran(3% w/v) is added (1 part dextran solution to 4 parts blood) and theblood allowed to stand for approximately 20 minutes at rt. Thesupernatant is carefully layered on a density gradient (Lymphoprep,Axis-Shield Healthcare) and centrifuged (15 mins, 2000 rpm, no brake).The supernatant is aspirated off and the cell pellet is re-suspended insterile saline (0.2%) for no longer than 60 seconds (to lysecontaminating red blood cells). 10 times volume of PBS is then added andthe cells centrifuged (5 mins, 1200 rpm). Cells are re-suspended inHBSS+(Hanks buffered salt solution (without phenol red) containingcytochalasin B (5 μg/mL) and 1 mM CaCl₂) to achieve 5×10⁶ cells/mL.

5×10⁴ cells are added to each well of a V-bottom 96 well plate and areincubated (30 mins, 37° C.) with the appropriate concentration of testcompound (0.3-1000 ng/mL) or vehicle (DMSO, 0.5% final conc).Degranulation is stimulated by addition of fMLP (final concentration 1μM). After a further incubation (30 mins, 37° C.), the cells are removedby centrifugation (5 mins, 1500 rpm) and the supernatants transferred toa flat bottom 96 well plate. An equal volume of tetramethylbenzidine(TMB) is added and, after 10 mins, the reaction terminated by additionof an equal volume of sulphuric acid (0.5 M) and absorbance read at 450nm (background at 655 nm subtracted). The 50% inhibitory concentration(IC₅₀) is determined from the resultant concentration-response curve.

(r) Cell Cytotoxicity Assay

1×10⁵ Jurkat cells (immortalised human T lymphocytes) are added to theappropriate number of wells of a 96 well plate in 100 μL of media (RPMIsupplemented with 10% foetal bovine serum). 1 μL of DMSO control (finalconcentration 1.0% v/v) or test compound (final concentration 20, 5 or 1μg/mL) is added to the wells and incubated at 37° C., 5% CO₂. After 24hours, the plate is centrifuged at 1200 rpm for 3 minutes and thesupernatant discarded. Cells are then resuspended in 150 μL (finalconcentration 7.5 μg/mL) of propidium iodide (PI) in PBS and incubatedat 37° C., 5% CO₂ for 15 minutes. After 15 minutes, cells are analysedby flow cytometry (BD accuri) using the FL3 window. The % viability iscalculated as the % of cells that are PI negative in the test wellsnormalised to the DMSO control.

In Vivo Screening: Pharmacodynamics and Anti-inflammatory Activity

(i) LPS-induced Neutrophil Accumulation in Mice

Non-fasted Balb/c mice are dosed by the intra tracheal route with eithervehicle, or the test substance at the indicated times (within the range2-8 hr) before stimulation of the inflammatory response by applicationof an LPS challenge. At T=0, mice are placed into an exposure chamberand exposed to LPS (7.0 mL, 0.5 mg/mL solution in PBS) for 30 min. Aftera further 8 hr, the animals are anesthetized, their tracheas cannulatedand BALF extracted by infusing and then withdrawing from their lungs 1.0mL of PBS via the tracheal catheter. Total and differential white cellcounts in the BALF samples are measured using a Neubauer haemocytometer.Cytospin smears of the BALF samples are prepared by centrifugation at200 rpm for 5 min at RT and stained using a DiffQuik stain system (DadeBehring). Cells are counted using oil immersion microscopy. Data forneutrophil numbers in BAL are represented as mean±S.E.M. (standard errorof the mean). The percentage inhibition of neutrophil accumulation iscalculated for each treatment relative to vehicle treatment.

(ii) Cigarette Smoke Model

A/J mice (males, 5 weeks old) are exposed to cigarette smoke (4%cigarette smoke, diluted with air) for 30 min/day for 11 days using aTobacco Smoke Inhalation Experiment System for small animals (ModelSIS-CS; Sibata Scientific Technology, Tokyo, Japan). Test substances areadministered intra-nasally (35 μL of solution in 50% DMSO/PBS) oncedaily for 3 days after the final cigarette smoke exposure. At 12 hrafter the last dosing, each of the animals is anesthetized, the tracheacannulated and bronchoalveolar lavage fluid (BALF) is collected. Thenumbers of alveolar macrophages and neutrophils are determined by FACSanalysis (EPICS® ALTRA II, Beckman Coulter, Inc., Fullerton, Calif.,USA) using anti-mouse MOMA2 antibody (macrophage) or anti-mouse 7/4antibody (neutrophil).

(iii) DSS-induced Colitis in Mice

Non-fasted, 10-12 week old, male BDF1 mice are dosed by oral gavagetwice daily with either vehicle, reference item (5-ASA) or test compoundone day before (Day −1) stimulation of the inflammatory response bytreatment with dextran sodium sulphate (DSS). On Day 0 of the study, DSS(5% w/v) is administered in the drinking water followed by BID dosing ofthe vehicle (5 mL/kg), reference (100 mg/kg) or test compound (5 mg/kg)for 7 days. The drinking water with DSS is replenished every 3 days.During the study, animals are weighed every day and stool observationsare made and recorded as a score, based on stool consistency. At thetime of sacrifice on Day +6, the large intestine is removed and thelength and weight are recorded. Sections of the colon are taken foreither MPO analysis, to determine neutrophil infiltration, or forhistopathology scoring to determine disease severity.

(iv) TNBS-induced Colitis in Mice

Non-fasted, 10-12 week old, male BDF1 mice are dosed by oral gavagetwice daily with either vehicle (5 mL/kg), reference item (Budesonide2.5 mg/kg) or test compound (1 or 5 mg/kg) one day before (Day −1)stimulation of the inflammatory response by treatment with2,4,6-trinitrobenzenesulphonic acid (TNBS) (15 mg/mL in 50% ethanol/50%saline). On Day 0 of the study TNBS (200 μL) is administeredintra-colonically via a plastic catheter with BID dosing of the vehicle,reference or test compound continuing for 2 or 4 days. During the study,animals are weighed every day and stool observations are made andrecorded as a score, based on stool consistency. At the time ofsacrifice on Day 2 (or Day 4), the large intestine is removed and thelength and weight recorded. Sections of the colon are taken forhistopathology scoring to determine disease severity.

(v) Adoptive Transfer in Mice

On Study day 0, female Balb/C mice are terminated and spleens obtainedfor CD45RB^(high) cell isolation (Using SCID IBD cell Separationprotocol). Approximately 4×10⁵ cells/mL CD45RB^(high) cells are theninjected intraperitoneally (100 μL/mouse) into female SCID animals. Onstudy day 14, mice are weighed and randomized into treatment groupsbased on body weight. On Day 14, compounds are administered BID, viaoral gavage, in a dose volume of 5 mL/kg. Treatment continues untilstudy day 42, at which point the animals are necropsied 4 hours afterthe morning administration. The colon length and weight are recorded andused as a secondary endpoint in the study as a measurement of colonoedema. The colon is then divided into six cross-sections, four of whichare used for histopathology scoring (primary endpoint) and two arehomogenised for cytokine analysis. Data shown is the % inhibition of theinduction window between nave animals and vehicle animals, where higherinhibition implies closer to the non-diseased, nave, phenotype.

(vi) Endotoxin-induced Uveitis in Rats

Male, Lewis rats (6-8 weeks old, Charles River UK Limited) are housed incages of 3 at 19-21° C. with a 12 h light/dark cycle (07:00/19:00) andfed a standard diet of rodent chow and water ad libitum. Non-fasted ratsare weighed, individually identified on the tail with a permanentmarker, and receive a single intravitreal administration into the rightvitreous humor (5 μL dose volume) of 100 ng/animal of LPS (Escherichiacoli 0111:B4 prepared in PBS, Sigma Aldrich, UK) using a 32-gaugeneedle. Untreated rats are injected with PBS. Test compound or vehicle(4% polyoxyl 40 stearate, 4% mannitol in PBS (pH 7.4)) are administeredby the topical route onto the right eye (10 μL) of animals 1 hour priorto LPS, at the time of LPS administration, and 1, 2 and 4 hours post LPSadministration. Before administration, the solution to be administeredis sonicated to ensure a clear solution. 6 hours after LPS dosing,animals are euthanized by overdose with pentobarbitone (via cardiacpuncture). Immediately after euthanasia, 10 μL of aqueous humor iscollected from the right eye of the rats by puncture of the anteriorchamber using a 32 gauge needle under a surgical microscope. The aqueoushumor is diluted in 20 μL of PBS and total cell counts are measuredimmediately using a Countess automated cell counter (Invitrogen).Following collection of the aqueous humour, the right eye of each animalis enucleated and dissected into front (anterior) and back (posterior)sections around the lens. Each section is weighed and homogenised in 500μL of sterile phosphate buffered saline followed by 20 minutescentrifugation at 12000 rpm at 4° C. The resulting supernatant isdivided into 3 aliquots and stored at −80° C. until subsequent cytokineanalysis by R&D DuoSet ELISA.

Summary of In Vitro and In Vivo Screening Results

TABLE 1 Dissociation constants for selected kinases determined byLeadHunter Discover Services (DiscoveRx Corporation, Fremont, CA), usingthe KINOMEscan ™ technology. Test Compound Dissociation Constant (nM)Example No. Lck p38 MAPKα Syk Example 1 4.2 2.8 7.1

Studies conducted by LeadHunter Discover Services (DiscoveRxCorporation, Fremont, Calif.) using the KINOMEscan™ technologydetermined that compound of Example 1 did not have any significanteffect on the binding of the kinases B-Raf and B-Raf (V600E) to theirstandard ligands. Moreover, this compound showed improved selectivitycompared to the Reference CompoundN-(4-(4-(3-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)ureido)naphthalen-1-yloxy)pyridin-2-yl)-2-methoxyacetamide(WO 2010/112936), as evidenced by lower selectivity scores (Table 1a).

TABLE 1a KinomeScan Selectivity score data at 50 and 500 nM; S(35) =(number of non-mutant kinases with % Ctrl <35)/(number of non-mutantkinases tested); S(10) = (number of non-mutant kinases with % Ctrl<10)/(number of non-mutant kinases tested); S(1) = (number of non-mutantkinases with % Ctrl <1)/(number of non-mutant kinases tested) KinomeScanSelectivity Scores/number of individual kinase hits 50 nM 500 nMCompound S(35) S(10) S(1) S(35) S(10) S(1) Reference 0.174/ 0.083/0.018/7 0.370/143 0.272/ 0.117/ Compound 67 32 105 45 Ex. 1 0.186/0.072/ 0.005/2 0.347/140 0.251/ 0.089/ 75 29 101 36

TABLE 1b Results from in vitro p38 MAPKα (Method 2), c-Src, Syk andGSK3α (Method 2) inhibition assays Test Compound IC50 Values for EnzymeInhibition (nM) Example No. p38 MAPKα c-Src Syk GSK3α 1 11 14 5 115

TABLE 2 Inhibition of cytokine release in stimulated cells (assays (b),(c) and (d) above). Test Compound PBMCs Example No. IL-8 IL-2 IFNγ 1 6.11125.5 9.5

As illustrated in Table 3 below, the compound of the example of thepresent invention is markedly less active than the Reference Compound(N-(4-(4-(3-(3-tert-butyl-1-p-tolyl-1H-pyrazol-5-yl)ureido)naphthalen-1-yloxy)pyridin-2-yl)-2-methoxyacetamide;WO 2010/112936) in assay (g) above, which measures impact on celldivision (mitosis) in PBMCs. Similarly, the compound of the example ofthe present invention is substantially less cytotoxic than the ReferenceCompound, displaying enhanced viabilities in cell cytotoxicity assay (r)above (Table 3).

TABLE 3 Effect of the compound of the invention on cell division inPBMCs (NT = not tested) and on Jurkat cell viability % Inhibition ofTest mitosis at % Viability % Viability % Viability compound 5 μg/mL at1 μg/mL at 5 μg/mL at 20 μg/mL Reference 87.8^(a) 23.5 18.9 17.3compound 1 2.4 94.6 96.1 95.8 ^(a)See, for example, the value reportedin WO 2013/050757.

As illustrated in Table 4 below, the compound of Example 1 significantlyand dose-dependently reduced cellular infiltration, as revealed bylowered cell counts, and cytokine IL-1β levels in both the anterior andposterior segments of the eyes of rats treated with intravitrealendotoxin LPS (see assay (vi) above).

TABLE 4 Dose-dependent effect of the compound of Example 1 on IL-1βlevels and cell counts in the eyes of LPS-stimulated rats. Data arereported as means ± SEM. IL-1β (pg/mL) IL-1β (pg/mL) Cell countsTreatment n Anterior tissue Posterior tissue (×10⁵/mL) Non-diseased 514.1 ± 6.3 30.8 ± 11.3  1.8 ± 0.2 Vehicle control 8 1636.6 ± 145.1 877.3± 115.6 69.9 ± 5.4 Example 1 8  367.3 ± 100.4 188.1 ± 54.7  21.9 ± 5.0(1 mg/mL) Example 1 8  791.2 ± 131.9 327.4 ± 61.4  30.4 ± 6.7 (0.1mg/mL) Example 1 8  980.0 ± 110.8 740.5 ± 56.2  43.5 ± 6.3 (0.01 mg/mL)Example 1 8 1558.1 ± 145.7 867.9 ± 120.8 63.6 ± 7.0 (0.001 mg/mL)Summary of Additional StudiesDetermination of Solubilities in Fasted-State Simulated Colonic Fluid(FaSSCoF)

The solubility of the compound of the invention in FaSSCoF at pH 6.5 isdetermined using a modification of a previously-reported procedure(Vertzoni, M., et al. Pharm. Res. 2010, 27, 2187-2196). In place of thebile salt extract employed in the original procedure (which extract isno longer available), the modified procedure uses a mixture of sodiumtaurochlorate (0.15 g), glycocholic acid (0.15 g), ursodeoxycholic acid(0.05 g), cholic acid (0.05 g), and glycodeoxycholic acid (0.05 g).These five bile acids are ground together with a mortar and pestle toproduce a fine white powder that is incorporated into the FaSSCoF, asoutlined below.

FaSSCoF Medium: Tris(hydroxymethyl)aminomethane (Tris; 0.275 g) andmaleic acid (0.44 g) are dissolved in water (35 mL) to give a solutionwhose pH is adjusted to 6.5 by treatment with 0.5M NaOH (ca. 12 mL). Thesolution is then made up to 50 mL with water. A portion of thisTris/maleate buffer solution (ca. 25 mL) is added to a 0.5 Lround-bottomed flask, before being treated with 0.00565 g of the bileacid mixture described above. Solutions of phosphatidylcholine (0.0111g) in DCM (0.15 mL) and palmitic acid (0.0013 g) in DCM (0.15 mL) areadded, then the organic solvent is evaporated off under reduced pressureat 40° C. until a clear solution, with no perceptible DCM odour, isachieved. The volume of the evaporated solution is adjusted to 50 mL byaddition of the remainder of Tris/maleate buffer, then BSA (0.115 g) isadded, before being dissolved by gentle agitation.

Solubility Determination: Test compounds are suspended in the pH 6.5FaSSCoF medium to give a maximum final concentration of 2-10 mg/mL. Thesuspensions are equilibrated at 25° C. for 24 h, before being filteredthrough a glass fibre C filter. The filtrates are then diluted asappropriate for injection and quantification by HPLC with reference to astandard. Different volumes of the standard, diluted and undilutedsample solutions are injected and the solubilities are calculated usingthe peak areas determined by integration of the peak found at the sameretention time as the principal peak in the standard injection.

FaSSCoF solubilities are shown in Table 5 below, which reveals that thecompound of the Example exhibited solubilities in the FaSSCoF medium atpH 6.5 of in excess of 0.01 mg/mL. For the compound of the Example ofthe present invention, pH 6.5 FaSSCoF solubilities were superior tothose of Reference Compound A,3-((4-((4-(3-(3-(tert-butyl)-1-(p-tolyl)-1H-pyrazol-5-yl)ureido)naphthalen-1-yl)oxy)pyrimidin-2-yl)amino)-5-ethynyl-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)benzamide{Fyfe, M. C. T., WO 2014/140582}.

TABLE 5 Solubilities measured for the compound of the Example of thepresent invention in FaSSCoF at pH 6.5. Test Compound pH 6.5 FaSSCoFSolubility (mg/mL) Example No. Run 1 Run 2 Run 3 Run 4 ReferenceCompound A <0.001 <0.001 1 0.016 0.018 0.010 0.011 1 (sodium salt) 0.0310.032 0.020 0.025 1 (hydrochloride salt) 0.016 0.015 — —Determination of Pharmacokinetic Parameters

Studies were conducted by Sai Life Sciences (Hinjewadi, Pune, India) toinvestigate the systemic pharmacokinetics and total colon tissuedistribution of the compound of the invention. In particular,pharmacokinetic studies were carried out in male C57BL/6 mice followinga single oral administration of the compound.

The data catalogued in Table 6 reveal that the compound of the inventionachieves substantial colonic concentrations, while, in contrast,systemic plasma or blood exposures are very low or negligible.

TABLE 6 Mean plasma or blood concentrations (ng/mL) or total colonlevels (ng/g) obtained following oral administration of the compound ofthe invention to mice at 5 mg/kg. Vehicle = 0.1% Tween 80 in 0.5%methylcellulose solution prepared in water. Cpd. Ex. No. Time (h) D M Mx0.5 1 2 4 6 8 12 24 1 1 PI 44.9 49.2 13.0 5.2 1.9 0.6 5.3 0.0 (Na salt)TC 4.3 9.3 52.4 10,403 4,049 12,898 535 41.4 Key to Table 6 D = compoundadministered (dosed) M = compound measured Mx = matrix PI = plasma Bd =blood TC = total colonhERG Inhibition Studies

The compound of the invention was tested for inhibition of the humanether a go-go (hERG) channel using IonWorks™ patch clampelectrophysiology at Essen Bioscience (Welwyn Garden City, England).

TABLE 7 hERG inhibition data for the compound of the invention %Inhibition at Top Top Example IC₅₀ (μM) Concentration Concentration1 >3.3 −4 3 μMAcute Eye Irritation/Corrosion Study

The objective of the acute eye irritation/corrosion study was to assessthe possible irritation or corrosion potential of the compound ofExample 1 at two selected dose levels (0.1 and 1.0 mg/mL), in comparisonto vehicle (4% w/v polyoxyethylene 40 stearate/4% w/v mannitol/phosphatebuffer (pH 7.4) solution), after one treatment day (Phase 1) or threeconsecutive treatment days (Phase 2) with four daily administrations(4-hours apart) by the ocular route (bi-lateral instillations of 40μL/eye/instillation) in the eyes of albino New Zealand White rabbits(13-15 weeks at initiation of dosing; 2 males and 2 females per dosegroup).

During the study, there were no unscheduled deaths, nor testitem-related clinical signs. Furthermore, there were no effects on bodyweight, nor on food consumption.

In Phase 1, ocular reactions were limited mainly to conjunctival redness(grade 1 or 2) and occasionally to chemosis (grade 1) and discharge(grade 1), in all groups, after instillation of the vehicle or of thetest item, Example 1, formulation at any dose levels. These scores wereslight to moderate. There were no differences in the frequency, severityand incidence between the Example 1-treated animals and the vehiclecontrols. The conjunctival redness was the most frequent reaction, andwas yet present (at grade 1) before the start of dosing. This localreaction is known to occur spontaneously in the albino rabbit duringocular studies and is related to the numerous ocular examinationsundergone on the animals. Chemosis and discharge were sporadicallyobserved in all groups after the first instillation and over the 3-dayobservation period thereafter. In addition, congestion of the iris wasoccasionally and unilaterally observed in the eyes of the two high dosegroup rabbits (1 mg/mL) and one vehicle group female. The Draizeexamination confirmed the integrity of the cornea after a singletreatment day and the photomotor reflex was normal for all animals onall occasions. In summary, the local tolerability of the formulationswas thus considered to be acceptable after a single dosing day. Similarlocal reactions were observed after instillation of the vehicle orformulations containing the compound of Example 1, indicative of amoderate vehicle-related effect on the ocular tolerance.

In Phase 2, the main ocular reactions were limited to conjunctivalredness (grade 1) in all groups after instillation of the vehicle or ofthe test item formulations at any dose levels. This score was slight andwas noted without any meaningful difference in the incidence andfrequency between groups throughout the 3-day treatment period. Theseverity was occasionally higher (grade 2) in the vehicle group than inthe test item-treated groups. This conjunctival redness was persistentand was still observed before the first instillation on the followingday. In addition, congestion of the iris was occasionally observed inthe eyes of the two high dose group rabbits and one vehicle groupfemale. No discharge was observed in any animals at all occasions. TheDraize examination confirmed the integrity of the cornea during the3-day treatment period. The photomotor reflex was normal for all animalson all occasions. In summary, the local tolerability of the formulationswas thus considered to be acceptable without any aggravation for the 3treatment days. Similar local reactions were observed after instillationof the vehicle alone or formulations containing the compound of Example1, indicative of the moderate vehicle-related effect on the oculartolerance.

Mutagenicity Assessment (Bacterial Reverse Mutation Screen)

Studies were conducted by Sequani (Ledbury, Herefordshire, UK) to assessthe compound of Example 1 in vitro for its ability to induce mutationsin four histidine dependent auxotrophic mutants of Salmonellatyphimurium, strains TA1535, TA1537, TA98 and TA100 and one tryptophandependent auxotrophic mutant of Escherichia coli, WP2 uvrA.

The mutation screen was conducted using the plate incorporation methodand was performed in both the presence and absence of S-9 mix (a liverpost-mitochondrial fraction derived from the livers of Aroclor 1254treated rats). The bacteria were exposed to the compound of Example 1dissolved in dimethylsulphoxide, which solvent was also used as thenegative control. The positive Control chemicals were Sodium Azide(TA1535 and TA100), 9-Aminoacridine (TA1537), 2-Nitrofluorene (TA98) and4-Nitroquinoline-N-Oxide (WP2 uvrA) in the absence of S-9 mix and2-Aminoanthracene (all strains) in the presence of S-9 mix.

The doses of the compound of Example 1 used in the mutation test underplate incorporation conditions were 15, 50, 150, 500 or 1500 μg/plate inall strains in the presence and absence of S-9 mix.

The compound of Example 1 was analysed up to the limit of solubility of1500 μg/plate in all strains in the presence and absence of S-9 mix,under plate incorporation conditions.

Precipitation was observed at 500 μg/plate in TA1537 and TA98 in thepresence of S-9 mix, and at 1500 μg/plate in all strains in the presenceand absence of S-9 mix. There was also a reduction in the mean colonycount at 500 μg/plate and 1500 μg/plate in TA98 and at 1500 μg/plate inTA1535, in the presence of S-9 mix, indicating toxicity of the test itemto the bacteria.

There were no dose-related or statistically significant increases inrevertant numbers observed in any strain at any dose level of thecompound of Example 1, in the presence or absence of S-9 mix, underplate incorporation conditions. This indicates the absence of anymutagenic effects for the compound of Example 1 under the conditions ofthe test.

Hydrolytic Stability Study

Chemical stability of the compound of the invention can be assessed in amixture of DMSO and water (3:1) at a test compound concentration of 1mg/mL.

General HPLC Procedure

-   -   Agilent, Waters X-Select C18, 2.5 μm, 4.6×30 mm column, 4 min        method, 5-95% MeCN/water (0.1% formic acid). Flow rate 2.5        mL/min. Column Oven Temperature 40° C. Detection 254 nm.

Sample Preparation

-   -   A 1.0 mg sample of test compound is dissolved in 750 μL of DMSO.        Water (250 μL) is added slowly, ensuring no precipitation        occurred.

Recording Stability

-   -   A 50 μL aliquot of the test solution is removed and analysed in        duplicate by 5 μL HPLC injections. The peak area for the test        compound is recorded following manual integration of the        corresponding UV trace.    -   The test solution is heated to 60° C., with stirring, and 50 μL        aliquots removed for HPLC analysis at 5 and 24 h timepoints. In        all cases, 5 μL injections are used and the samples analysed in        duplicate.    -   The peak areas for the test compounds are recorded at both        subsequent timepoints and the % decomposition calculated from        the % change in peak area over time.    -   Reference Compound B        (3-ethynyl-5-((4-((4-(3-(3-isopropyl-1-(p-tolyl)-1H-pyrazol-5-yl)ureido)naphthalen-1-yl)oxy)pyrimidin-2-yl)amino)-N-(2-morpholinoethyl)benzamide;        Cariou, C. A. M., et al, WO 2014/027209) is included in each        stability study as a control to validate the study.        Stability of Pharmaceutical Formulations

20 mL of 1 mg/mL stock solutions were prepared, in duplicate, of thesodium (Na) and hydrochloride (HCl) salts of the compound of Example 1as follows: The appropriate quantities of each salt were mixed with 10mM pH 7.2 phosphate buffer containing 4.5% mannitol and 3% polyoxyl 40stearate. The samples were sonicated to achieve clear solutions havingthe following properties: Osmolality (mOsm/kg): 310 (Na), 314 (HCl); pH:7.00 (Na), 7.05 (HCl). 0.5 mL of the stock solutions were diluted to 1mL with 20% DMSO in water & injected for purity analysis by HPLC. Theremaining stock solutions were then split into aliquots of 0.5 mL inHPLC vials, and stored at various conditions in duplicate. Samples werestored at 5 and 25° C., before being analysed by HPLC at 1, 2 and 4weeks. Separate samples were stored at 40° C. and analysed at 4 weeks.The analysis shown in the Table below reveals that the Compound ofExample 1 is stable in solution at 5° C.

Original Purity Purity (%) at week n Test substance Sample (%) Temp (°C.) n = 1 n = 2 n = 4 Example 1, 1 98.6 5 98.2 98.4 98.3 sodium salt 2598.4 98.1 97.6 40 — — 76.0 2 98.5 5 98.4 98.5 98.4 25 98.5 98.1 97.6 40— — 73.7 Example 1, 1 98.1 5 98.2 98.1 97.9 hydrochloride salt 25 98.097.7 97.2 40 — — 79.2 2 98.1 5 98.2 98.0 98.0 25 98.0 97.6 97.4 40 — —80.4Abbreviations

-   AcOH glacial acetic acid-   aq aqueous-   5-ASA 5-aminosalicylic acid-   ATP adenosine-5′-triphosphate-   BALF bronchoalveolar lavage fluid-   BID bis in die (twice-daily)-   BINAP 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl-   BOP (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium    hexafluorophosphate-   br broad-   BrdU 5-bromo-2′-deoxyuridine-   BSA bovine serum albumin-   CatCart® catalytic cartridge-   CDI 1,1-carbonyl-diimidazole-   COPD chronic obstructive pulmonary disease-   d doublet-   dba dibenzylideneacetone-   DBU 1,8-diazabicyclo[5.4.0]undec-7-ene-   DCC dicyclohexylcarbodiimide-   DCM dichloromethane-   DIAD diisopropyl azodicarboxylate-   DIPEA diisopropylethylamine-   DMAP 4-dimethylaminopyridine-   DMEM Dulbecco's modified eagle medium-   DMF N,N-dimethylformamide-   DMSO dimethyl sulfoxide-   DPPA diphenylphosphoryl azide-   d-U937 cells PMA differentiated U-937 cells-   EDTA ethylenediaminetetraacetic acid-   ELISA enzyme-linked immunosorbent assay-   (ES⁻) electrospray ionization, negative mode-   (ES⁺) electrospray ionization, positive mode-   Et ethyl-   Et₃N triethylamine-   EtOAc ethyl acetate-   EtOH ethanol-   FACS fluorescence-activated cell sorting-   FBS foetal bovine serum-   FCS foetal calf serum-   fMLP formyl-methionyl-leucyl-phenylalanine-   FRET fluorescence resonance energy transfer-   GSK3α glycogen synthase kinase 3α-   HBEC primary human bronchial epithelial cells-   HBSS Hank's balanced salt solution-   HPLC high performance liquid chromatography-   HPMC hydroxypropylmethylcellulose-   h or hr hour(s)-   HATU 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium    hexafluorophosphate-   HOAt 1-hydroxy-7-azabenzotriazole-   HOBt hydroxybenzotriazole-   HRP horseradish peroxidise-   HRV human rhinovirus-   ICAM-1 inter-cellular adhesion molecule 1-   IFNγ interferon-γ-   IL interleukin-   iPrOAc isopropyl acetate-   JNK c-Jun N-terminal kinase-   LC liquid chromatography-   Lck lymphocyte-specific protein tyrosine kinase-   LPS lipopolysaccharide-   m multiplet-   (M+H)⁺ protonated molecular ion-   MAPK mitogen-activated protein kinase-   MAPKAP-K2 mitogen-activated protein kinase-activated protein    kinase-2-   mCPBA meta-chloroperbenzoic acid-   Me methyl-   MeCN acetonitrile-   MeOH methanol-   MHz megahertz-   min or mins minute(s)-   MMAD mass median aerodynamic diameter-   MOI multiplicity of infection-   MPO myeloperoxidase-   MIT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-   MS mass spectrometry-   m/z mass-to-charge ratio-   NMP N-methyl pyrrolodinone-   NMR nuclear magnetic resonance (spectroscopy)-   OD optical density-   PBMC peripheral blood mononuclear cell-   PBS phosphate buffered saline-   Ph phenyl-   PHA phytohaemagglutinin-   PMA phorbol myristate acetate-   pTSA 4-methylbenzenesulfonic acid (para-toluenesulfonic acid)-   PyBOP (benzotriazol-1-yloxy)tripyrrolidinophosphonium    hexafluorophosphate-   q quartet-   rt or RT room temperature-   RP HPLC reverse phase high performance liquid chromatography-   rpm revolutions per minute-   RPMI Roswell Park Memorial Institute-   RSV respiratory syncytial virus-   s singlet-   sat or satd saturated-   SCID severe combined immunodeficiency-   SCX solid supported cation exchange (resin)-   SDS sodium dodecyl sulfate-   S_(N)Ar nucleophilic aromatic substitution-   Syk Spleen tyrosine kinase-   t triplet-   T3P 1-propanephosphonic acid cyclic anhydride-   TBAF tetrabutylammonium fluoride-   TBDMS tert-butyldimethylsilyl-   TCID₅₀ 50% tissue culture infectious dose-   TEA triethylamine-   THF tetrahydrofuran-   TFA trifluoroacetic acid-   TGFβ transforming growth factor beta-   TIPS triisopropylsilyl-   TMB 3,3′,5,5′-tetramethylbenzidine-   TMS-Cl trimethylsilyl chloride-   TNFα tumor necrosis factor alpha

Prefixes n-, s-, i-, t- and tert- have their usual meanings: normal,secondary, iso, and tertiary.

What is claimed is:
 1. A compound of formula VIIa,

wherein Q^(x) represents —C(O)O—C₁₋₄ alkyl, or a salt thereof.
 2. Acompound of formula VIIa, or a salt thereof, as claimed in claim 1,wherein said compound is methyl4-((4-((4-(3-(5-(tert-butyl)-2-methoxy-3-(methylsulfonamido)phenyl)ureido)naphthalen-1-yl)oxy)pyridin-2-yl)amino)-2-methoxybenzoate.3. A process for the preparation of a compound of formula VIIa, asdefined in claim 1, which process comprises: (a) reaction of a compoundof formula IIp,

with a compound of formula IIIp,

wherein one of Z^(1p) and Z^(2p) is a structural fragment of formula IV,

and the other of Z^(1p) and Z^(2p) is a structural fragment of formulaVp

wherein Q^(x) represents —C(O)O—C₁₋₄ alkyl; (b) reaction of a compoundof formula IIap

 wherein Z^(1p) is as defined above, with a suitable azide-formingagent,  which reaction is followed, without isolation, by thermalrearrangement of the intermediate acyl azide (of formula Z^(1p)—C(O)—N₃)to provide, in situ, a compound of formula II, which compound is thenreacted with a compound of formula IIIp as defined above; (c) reactionof a compound of formula IIbp,

 wherein LG¹ represents a leaving group and Z^(1p) is as defined above,with a compound of formula IIIp, as defined above; or (d) reaction of acompound of formula VI,

wherein LG² represents a leaving group, with a compound of formula VIIp,

wherein Q^(x) is as defined above.